Induction of hypoglycaemia in Japanese encephalitis virus infection: the role of T lymphocytes

We report here development of hypoglycaemia in the convalescent phase of Japanese encephalitis virus (JEV) infection in mice by the induction of antigen-specific Ly1⁻2⁺ T cells in the spleen which mediate hypoglycaemia through the generation of soluble T cell hypoglycaemic factor (TCHF). The TCHF acted in a dose-dependent manner and was found to be trypsin-sensitive and thermolabile. It was purified on Superose-12 high performance liquid chromatography (HPLC) gel filtration column and purified protein migrated as a ~25-kD band on SDS–PAGE. The JEV-induced hypoglycaemia coincided with an increased circulating glucagon level, without any alterations in blood insulin and growth hormone concentrations. These effects were mimicked by TCHF. These results indicate that JEV-primed T lymphocytes mediate hypoglycaemia through the production of a soluble hypoglycaemic factor.     

Correlation of tumor necrosis factor levels in the serum and cerebrospinal fluid with clinical outcome in Japanese encephalitis patients

To investigate the prognostic role of tumour necrosis factor (TNF) in Japanese encephalitis virus (JEV) infection, we measured the immunoreactive forms of TNF concentrations in the serum and cerebrospinal fluid (CSF) of 47 laboratory-confirmed cases of JE. It was observed that TNF levels were elevated (>15 pgm/ml) in all the 47 serum samples (range 19.4–923.8 pg/ml), while in 46/47 CSF samples TNF was elevated (range 10.8–376 pg/ml). The mean (SD) TNF levels in the serum of fatal cases was 234.34 pg/ml (304.40) as compared to the mean of 85.31 pg/ml (SD 153.92) in nonfatal cases. Similar observations were also made with respect to the TNF levels in the CSF; the mean of fatal cases was 69.39 pg/ml (SD 39.00) in contrast to the mean of 62.41 pg/ml (SD 75.25) of nonfatal cases. The increase in TNF levels did not show any correlation to the duration of illness. It was further observed that the mortality rate increased with increasing concentrations of TNF in the serum and CSF. Correlation of laboratory parameters to final outcome revealed that TNF concentrations above 50 pg/ml in serum correlated significantly (P = .05) with a fatal outcome, whilst high levels of JEV-IgM antibodies (>500 units) in the CSF correlated with a nonfatal outcome (P = .03). These results suggest that TNF can be used as a possible prognosticator of a fatal outcome in JEV infection. J Med Virol 51:132–136, 1997. © 1997 Wiley-Liss, Inc.     

逆转录-聚合酶链反应方法检测乙型脑炎病人标本

目的 逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）检测乙型脑炎（乙脑）病人标本方法的建立和评估。方法 建立ＲＴ－ＰＣＲ法,了解该方法用于乙脑病毒检测的敏感性,特异性,并用于临床疑似乙脑病人血清及脑脊液（ＣＳＦ）标本的检测,并与反向被动血凝抑制实验（ＲＰＨＩ）方法进行比较分析。结果 用该ＲＴ－ＰＣＲ法检测高顺生株（高株）敏感性可达６４ＰＦＵ。共检测临床疑似乙脑病人标本３８份,对ＣＳＦ中乙型脑炎病毒（ＪＥＶ）     

224例流行性乙型脑炎流行特征分析

目的分析224例流行性乙型脑炎（简称乙脑）的流行特征,为进一步做好乙脑的预防控制措施提供科学依据。方法采用快速IgM抗体捕捉ELISA进行患者血清或脑脊液的乙脑抗体检测,结合临床表现予以诊断。对确诊病例利用Excel软件进行描述流行病学分析。结果 2001-2010年共收集疑似乙脑患者血清/脑脊液标本626份,阳性224例,检出率为35.78%,脑脊液的阳性检出率略高于血清。男女比例为1.24：1,好发年龄为10岁以下及50岁以上,发病者多数在农村。发病时间7-10月,发病高峰集中在8月。结论开展乙脑病例监测,在高发地区及农村进行重点控制,加强宣传教育和增强灭蚊、防蚊意识,加强中老年人的乙脑疫苗免疫接种,是降低乙脑发病率的必要措施。     

Replicon-based Japanese encephalitis virus vaccines elicit immune response in mice

In this study, a replicon vaccine vector system for Japanese encephalitis virus (JEV) was established. The system included a trans-complementing cell line, a series of JEV DNA-based subgenomic replicons, and several encapsidated JEV propagation-deficient pseudoinfectious particles (PIPs). The DNA-based JEV replicon vectors, which deleted the structural coding region, could be able to self-replicate and express the reporter gene. A stable BHK packaging cell line named BHK-CME, which constitutively expressed the capsid protein C, the precursor membrane and envelope proteins (C-prM-E) of JEV, was generated. BHK-CME cells were used to trans-complement the JEV replicons and proved to package the JEV replicons into single-round infectious PIPs efficiently. The PIPs were produced in titers of up to 1.6 × 10⁵ IU/ml. To investigate the efficacy of JEV replicon-based vaccines, four groups of female BALB/c mice were inoculated three times at 3-week intervals with the JEV PIPs and others. The JEV-specific antibody titers reached to 1:6400 and the neutralizing antibody titers reached 1:256 after three rounds of immunization with JEV PIPs. And the antisera collected from immunized mice were shown to be protective partially against lethal infection when passively transferred to susceptible weanling mice. These results demonstrated the value of the JEV replicon vector system for the development of new vaccine candidates.     

Japanese encephalitis virus latency in peripheral blood lymphocytes and recurrence of infection in children.

In a study group of 40 children who had been admitted to hospital with acute encephalitis, the disease was due to infection with Japanese encephalitis virus (JEV). Three children developed recurrence of disease 8-9 months later. No virus had been isolated from these three patients during the acute stage of their illness, but virus was recovered from all during the recurrence phase by co-cultivation of their peripheral blood mononuclear cells in primary mouse embryo fibroblast cultures. Virus was also recovered by co-cultivation of peripheral blood mononuclear cells collected 8 months after their acute disease from three out of eight randomly selected asymptomatic children within the study group but not from similar cultures set up from JEV-seronegative children used as controls. Virus was also isolated by co-cultivation of T lymphocytes of asymptomatic children as detected by indirect immunofluorescence or by inoculation in mice.     

Effect of macrophage-derived factor on hypoferraemia induced by Japanese encephalitis virus in mice.

Depression of serum iron following Japanese encephalitis virus (JEV) infection was observed in mice. The hypoferraemia was associated with the accumulation of iron in reticulo-endothelial cells in the spleen. Splenectomy (compared with sham-operation) prevented the depression in serum iron concentration after JEV infection. It also prevented the rise in levels of liver iron. The effect of JEV-stimulated, splenic macrophage-derived factor (MDF) was evaluated in causing hypoferraemia. MDF produced a rapid reduction in the serum iron levels with accumulation of iron in spleen. These observations suggest that MDF plays a key role in the regulation of iron metabolism during JEV infection.     

Neutrophil chemotactic factor produced by Japanese encephalitis virus stimulated macrophages.

The mechanism of neutrophil leucocytosis in cases of Japanese encephalitis is not known. We here report that during Japanese encephalitis virus (JEV) infection in mice the splenic macrophages secrete a chemotactic factor that attracts the neutrophils. The peak activity of macrophage derived factor (MDF) was observed on day 7 following infection. The MDF acted in a dose-dependent manner. This chemoattractant was purified by low pressure liquid chromatography and gave a single band of 10 kD on silver stained polyacrylamide gel. The MDF was found to be heat resistant and sensitive to prolonged incubation with proteases.     

Determining the clinical characteristics and prognostic factors for the outcomes of Japanese encephalitis in adults: A multicenter study from southern Taiwan

BackgroundIn Southeast Asia, Japanese encephalitis (JE) is an important cause of viral encephalitis which may cause severe neurological sequelae. JE affects mostly children; therefore, clinical presentations and prognosis of adult JE patients are seldom addressed. This study aimed to describe the clinical characteristics and prognostic factors for the outcome of adult JE patients.MethodsMedical records of adult JE patients with acute encephalitis syndrome during 2001–2018 from five medical centers in southern Taiwan were reviewed. Clinical characteristics, brain images, and prognostic factors for outcomes were analyzed. Patients were divided into the good outcome (GO) group and poor outcome (PO) group according to their Glasgow Coma Scale (GCS) scores (GCS >8 vs. ≤ 8) at discharge.ResultsSixty-eight patients (men, 61.8%; median age, 50 years) were included. Summer is the epidemic season, and the number of cases peaked in June. The most common symptoms at initial presentation were altered consciousness and fever (both 94.1%), followed by headache (51.4%). The most commonly involved brain regions were thalamus (55.7%) and basal ganglion (37.7%). The median GCS score at nadir was 8, and the median time from onset to nadir was five days. Fifty-two patients were included in the GO group, while 16 were included in the PO group. On multivariate analysis, flaccidity, rigidity, and elevated CSF protein level were identified as independent prognostic factors for PO.ConclusionInitial clinical presentations of abnormal muscle tone including flaccidity, rigidity and high CSF protein levels are independent prognostic factors for PO in adult JE patients.     

Field trial of a Japanese encephalitis diagnostic kit

Serum and cerebrospinal fluid (CSF) obtained from patients in rural Thailand during an encephalitis epidemic were assayed with a Japanese encephalitis rapid diagnosis kit. Japanese encephalitis was diagnosed by detection of virus-specific IgM (JEV IgM) in CSF (1:10 dilution) or serum (1:100 dilution) with an antibody capture enzyme-linked immunosorbent assay. Specimens were assayed immediately on site at the provincial hospital and scored by visual examination within 4 h. Each specimen was retested carefully later to accurately determine its activity (units) at a single screening dilution; each was tested also at serial dilutions to determine its end-point titer. On-site kit results showed close agreement with subsequent laboratory results for detection and quantitation of JEV IgM and JEV IgG in either serum or CSF. Using the kit on site, admission CSF from 35 (73%) of 48 laboratory-proven JEV-infected patients were scored as definitely positive for JEV IgM, while all 17 CSF specimens from non-JEV infected patients were read as negative (sensitivity 73%, specificity 100%). A rapid and early diagnosis of acute Japanese encephalitis can be accomplished almost anywhere.     

Antiviral effect of nitric oxide during Japanese encephalitis virus infection

The ability of Japanese encephalitis virus (JEV) and JEV-induced macrophage derived neutrophil chemotactic factor (MDF) to produce nitric oxide (NO), and the possible antiviral effect of NO during JEV infection, was investigated. Splenic macrophages of JEV infected mice produced maximum NO in vivo at day 7 post infection, and in vitro at 24 h after JEV stimulation. MDF-induced NO production was dose dependent and maximal at 60 min after MDF treatment. The response was sensitive to anti-MDF antibody treatment and the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). Pretreatment of mice with L-NMMA increased the mortality to 100% in JEV infected mice in vivo and inhibited NO production in vitro, while MDF stimulated macrophages inhibited virus replication with high levels of NO production. MDF treatment increased the survival rate of JEV infected mice. The findings thus demonstrate that MDF induces production of NO during JEV infection, which has an antiviral effect. This may be one of the important mechanisms of natural immunity in controlling the initial stages of JEV infection.     

Experimental evidence that RNA recombination occurs in the Japanese encephalitis virus

Due to the lack of a proofreading function and error-repairing ability of genomic RNA, accumulated mutations are known to be a force driving viral evolution in the genus Flavivirus, including the Japanese encephalitis (JE) virus. Based on sequencing data, RNA recombination was recently postulated to be another factor associated with genomic variations in these viruses. We herein provide experimental evidence to demonstrate the occurrence of RNA recombination in the JE virus using two local pure clones (T1P1-S1 and CJN-S1) respectively derived from the local strains, T1P1 and CJN. Based on results from a restriction fragment length polymorphism (RFLP) assay on the C/preM junction comprising a fragment of 868 nucleotides (nt 10-877), the recombinant progeny virus was primarily formed in BHK-21 cells that had been co-infected with the two clones used in this study. Nine of 20 recombinant forms of the JE virus had a crossover in the nt 123-323 region. Sequencing data derived from these recombinants revealed that no nucleotide deletion or insertion occurred in this region favoring crossovers, indicating that precisely, not aberrantly, homologous recombination was involved. With site-directed mutagenesis, three stem-loop secondary structures were destabilized and re-stabilized in sequence, leading to changes in the frequency of recombination. This suggests that the conformation, not the free energy, of the secondary structure is important in modulating RNA recombination of the virus. It was concluded that because RNA recombination generates genetic diversity in the JE virus, this must be considered particularly in studies of viral evolution, epidemiology, and possible vaccine safety.     

广西流行性乙型脑炎50年流行趋势分析

目的分析广西乙脑流行趋势和特征,掌握其流行规律,为制定防治对策提供科学依据。方法采用描述性流行病学分析方法回顾性统计广西全区1960-2010年乙脑疫情资料。结果 1960-2010年全区共发生乙脑65384例,死亡11875例,年均发病率为3.38/10万,年均死亡率为0.61/10万,病死率为18.16%;发病流行以1960年代和1970年代为重,年均发病率分别为8.59/10万和9.24/10万,20世纪70年代以后广西开始持续推广使用乙脑疫苗,包括流行季节前突击接种和纳入免疫规划管理,1980-2000年代的年均发病率分别为1.75/10万,1.34/10万,0.44/10万;全区所有县和市辖区都有病例发生,但病例主要集中于桂西北、桂南和桂东地区,这些地区为乙脑的高发区;发病主要集中于5-9月份;患者以0～14岁农村散居儿童为主。结论广西乙脑发病流行呈下降趋势,发病有明显季节性;发病主要集中在低年龄儿童;经过持续多年的乙脑疫苗突击接种和纳入免疫规划管理,广西乙脑防控已取得明显成效。     

中国基因3型乙型脑炎病毒E基因分子特征

目的 以减毒活疫苗(SA14-14-2株)为对照,分析我国分离的基因3型乙脑病毒E基因区段核苷酸及氨基酸序列分子特征.方法 从GenBank中获取相应乙脑病毒株E基因区段核苷酸序列,通过Clustal X(1.81)、DNAStar、GENEDOC(3.2)等生物学软件进行核苷酸和氨基酸位点差异分析.以蜱传脑炎病毒可溶性蛋白晶体结构为模板进行乙脑病毒E蛋白氨基酸位点分析.结果 我国不同地域、不同宿主分离的基因3型乙脑病毒与SA14-14-2株核苷酸同源性分别在96%和95%以上,氨基酸同源性在95%和94%以上.在同一地域、同一宿主类型分离的毒株之间核苷酸和氨基酸同源性非常高.在E基因区段存在10处共同的氨基酸位点差异,在结构域Ⅰ(E160)、结构域Ⅱ(E123和E227)和两个未在结构域中的氨基酸位点(E441和E487)等5个位点在部分基因3型乙脑病毒中存在差异.结论 我国分离的基因3型乙脑病毒与减毒活疫苗株(SA14-14-2株)E基因区段同源性高,存在5处基因3型乙脑病毒特异的氨基酸位点差异,但现行减毒活疫苗株理论上可以保护我国分离的基因3型乙脑病毒野毒株.     

Immunomodulatory cytokines determine the outcome of Japanese encephalitis virus infection in mice

Japanese encephalitis virus (JEV) induces an acute infection of the central nervous system, the pathogenic mechanism of which is not fully understood. To investigate host response to JEV infection, 14‐day‐old mice were infected via the extraneural route, which resulted in encephalitis and death. Mice that received JEV immune splenocyte transfer were protected from extraneural JEV infection. Pathology and gene expression profiles were then compared in brains of mice that either succumbed to JEV infection or were protected from infection by JEV immune cell transfer. Mice undergoing progressive JEV infection had increased expression of proinflammatory cytokines, chemokines, and signal transducers associated with the interferon (IFN) pathway. In contrast, mice receiving immune cell transfer had increased production of the Th2 cytokine IL‐4, and of IL‐10, with subdued expression of IFN‐γ. We observed IL‐10 to be an important factor in determining clinical outcome in JEV infection. Data obtained by microarray analysis were further confirmed by quantitative RT‐PCR. Together, these data suggest that JEV infection causes an unregulated inflammatory response that can be countered by the expression of immunomodulatory cytokines in mice that survive lethal infection. J. Med. Virol. 82:304–310, 2010. © 2009 Wiley‐Liss, Inc.     

Japanese encephalitis in a Finnish traveler on a two-week holiday in Thailand

Japanese encephalitis (JE) virus is a mosquito-borne flavivirus, and one of the leading causes of epidemic encephalitis in Southeast Asia. Reports of symptomatic JEV encephalitis in tourists have been rare. We describe a case of symptomatic JE transmitted in 2004 during a short two-week trip to common tourist attractions in Thailand.     

四川省分离的基因1型乙型脑炎病毒分子特征分析

目的 从四川省巴中市采集的蚊虫标本中分离乙型脑炎(简称乙脑)病毒(JEV),确定其基因型别,并分析相关的基因1型乙脑病毒PrM和E基因区段氨基酸序列特征.方法 对2004年采集蚊虫标本进行病毒分离,对新分离的乙脑病毒进行生物学、血清学及分子生物学鉴定.逆转录聚合酶链反应(RT-PCR)扩增新分离JEV的PrM、E区段核苷酸序列,测序后应用Clustal X软件做碱基配对分析,MEGA4软件完成病毒进化分析,GENEDOC(3.2)软件完成氨基酸位点分析,根据蜱传脑炎病毒可溶性蛋白晶体结构为模板进行乙脑病毒E蛋白三维结构模拟预测分析.结果 共采集4668只蚊虫标本,主要是骚扰阿蚊和库蚊,分离到6株病毒,经鉴定均属于基因1型的乙脑病毒.将四川省分离的6个毒株结合我国新分离的基因1型乙脑病毒与减毒活疫苗株SA14-14-2株的PrM区段和E区段氨基酸比较,发现PrM区段在PrM2、64和65位存在基因1型乙脑病毒独有的氨基酸位点差异,E区段存在14处共同的氨基酸位点差异,其中在E129、222、327和366位点为中国目前分离到的基因1型乙脑病毒所特有的位点特征.结论 从四川省巴中市首次分离到基因1型的乙脑病毒,并发现基因1型乙脑病毒与减毒活疫苗株之间PrM、E基因区段存在氨基酸差异,但现行疫苗株理论上可以保护新分离的基因1型乙脑病毒.     

Antibody-capture ELISA for detection of immunoglobulin M antibodies in sera from Japanese encephalitis and dengue hemorrhagic fever patients

Immunoglobulin M (IgM) antibody titers in paired sera from 19 encephalitis and 44 dengue hemorrhagic fever (DHF) patients in Thailand and 42 Japanese encephalitis (JE) patients in Japan were measured by the antibody capture ELISA and applied to distinguish JE virus infection from dengue virus infection. Titer distribution and the ratio of the titers against JE and dengue antigens led to the following diagnostic criteria. The specimens can be considered as positive with JE when IgM-ELISA titer showed over 200 against JE and 4-fold or more higher than titers against any types of dengue antigens. The specimens can be considered as positive with dengue infection when IgM ELISA titer showed over 200 against one of the 4 types of dengue antigens and 4-fold or more higher than against JE antigen. Based on these criteria, 41 of 42 patients in Japan and 11 of 19 encephalitis patients in Thailand could be diagnosed as having JE virus infection while 2 of 19 encephalitis patients in Thailand and 26 of 44 DHF patients in Thailand could be diagnosed as having dengue virus infections.