Epidemic Japanese B encephalitis combined with contactinassociated protein-like 2 antibody-positive autoimmune encephalitis:A case report

BACKGROUND It is not uncommon to develop viral encephalitis.Epidemic Japanese B encephalitis infection combined with contactin-associated protein-like 2(CASPR-2)antibody-positive autoimmune encephalitis has not been reported at present.In clinical work,we need to consider more options.CASE SUMMARY A 32-year-old male worker presented with headache,fever and call-unresponsive presentation.Complete cranial magnetic resonance image showed symmetrical abnormal signals in bilateral medial temporal lobe,bilateral thalamus and basal ganglia.Improved lumbar puncture showed that cerebrospinal fluid protein and cell count increased significantly.Viral encephalitis was considered,and the patient's consciousness still increased rapidly after antiviral treatment.Further detection of Cerebrospinal fluid Japanese B encephalitis virus Polymerase Chain Reaction positive,serum autoimmune encephalitis antibody showed CASPR-2 antibody positive(1:320),the patient's condition gradually improved after plasma exchange treatment.3 mo later,the serum CASPR-2 antibody was negative and the patient's condition was stable.CONCLUSION This article reports the world’s first case of Epidemic Japanese B encephalitis infection combined with CASPR-2 antibody-positive autoimmune encephalitis,with a view to raising awareness.

Agranulocytosis following injection of inactivated Japanese encephalitis vaccine(Vero cell):A case report

BACKGROUND Japanese encephalitis virus(JEV),a mosquito borne flavivirus,is the leading cause of viral encephalitis in Asia,in terms of frequency and severity.JEV infection is thought to confer lifelong immunity.With the near eradication of poliomyelitis,JEV is now the continent’s leading cause of childhood viral neurologic infection and disability.The most common clinical manifestation of JEV infection is acute encephalitis,and currently there is no specific antiviral therapy.Japanese Encephalitis Vaccine(JE-VC)is an effective prevention measure,including JE-VC,Live(JE-MB),and Inactivated JE-VC.CASE SUMMARY A 9-mo-old girl received injection of Inactivated JE-VC(Vero cell)(Liaoning Chengda,batch number 201611B17)on August 31,2017.On that night,she developed a fever with the body temperature up to 38.5°C,for which Ibuprofen Suspension Drops 1.25 mL was given as antipyretic treatment.On September 1,the patient developed apocleisis,and her parents noticed herpes in her oral cavity.The patient was sent to our hospital on September 3.Physical examination led to a diagnosis of herpetic stomatitis,for which Stomatitis Spray 1 puff,tid,Kangfuxin Liquid 2 mL,tid,and vitamin B20.5 tablet,tid,were prescribed.Routine blood tests for low fever on September 6,2017 revealed an absolute neutrophil count(ANC)of 0.62×109/L,hemoglobin(Hb)of 109 g/L,and platelet count(PLT)of 308×10^(12)/L,and the tests were monitored regularly thereafter.The patient was followed until July 26,2020,when routine blood tests revealed ANC 1.72×109/L,Hb 138 g/L,and PLT 309×1012/L,indicating that the neutropenia count had normalized.CONCLUSION This report attempts to bring to clinical attention that Inactivated JE-VC(Vero cell)might cause prolonged granulocytopenia or even agranulocytosis.

A retrospective investigation on the phenotype and stability of the E-protein gene in Japanese Encephalitis (JE) virus strain SA14-14-2 used live-attenuated JE vaccine

To detect retrospectively the phenotype and stability of the E-protein gene in Japanese Encephalitis (JE) virus strain SA14-14-2 used in the live-attenuated JE vaccine prepared at different years, the viral titer was titrated by plaque formation in BHK-21 cell cultures, and the neuro-virulence of viruses was assayed in mice with body weight of 12-14 g by intracerebral inoculation. Meanwhile, the total RNA of virus gene was extracted and amplified by RT-PCR with the designed primers, and then it was purified and cloned to the expression vector pGEM-T. The recombinant plasmid was purified and sequenced. It was found that the loss of viral titer of vaccines stored in -20℃ for longer than 10 years was less than 0.5 Lg PFU/ml. No mice inoculated intracerebrally showed signs of illness or even death. The size of plagues of the vaccine virus remained to be small, and the E genes of primary virus seed SA14-14-2 and the vaccines prepared at different years (1987-2001) were unchanged, including the 8 critical amino acid sites which were different from the parent wild virus strain SA14 and the related neuro-virulence. These results indicate that the genotypic and biological characteristics of the attenuated JE virus strain SA14-14-2 and its vaccines prepared are quite stable without any reversion noted.

JEV、PRRSV、PPV及PRV多重PCR检测方法的建立及应用

为了建立一种能快速鉴别猪日本脑炎病毒(JEV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪细小病毒(PPV)和猪伪狂犬病病毒(PRV)的准确、高效的多重常规PCR检测方法,针对JEV、PRRSV、PPV、PRV基因保守区域合成特异性引物,优化后获得最优反应条件,对该方法特异性、敏感性、重复性进行了测定,并应用该方法对临床样品进行初步应用。结果显示,该方法对JEV、PRRSV、PPV、PRV可进行特异性扩增,对混合阳性质粒检测下限达2×10^(6)copies/μL,对猪圆环病毒2型、猪瘟病毒、猪流行性腹泻病毒、猪丁型冠状病毒、猪传染性胃肠炎病毒等相关病毒均无扩增,且重复性良好。用该方法检测51份2021年采集的广西区内组织样品,检出JEV、PRRSV、PPV、PRV阳性率分别为7.84%、50.98%、5.88%、23.53%,且存在多重混合感染的情况。所建立的多重PCR方法具有良好的特异性、敏感性及重复性,可应用于临床常见猪繁殖障碍性病毒病的快速诊断和监测预警,为猪繁殖障碍性病毒病提供了诊断技术支持。

Mechanism of Japanese encephalitis virus genotypes replacement based on human,porcine and mosquito-originated cell lines model

Objective:To examine the multiplication efficiency Japanese encephalitis virus(JEV)genotype Ⅰ(G Ⅰ) and genotype Ⅲ(GⅢ) of different cell lines which originated from human,porcine,mosquitoes in order to prove mechanism of JEV G Ⅰ replacement JEV GⅢ since it emerging in nature recent decades.Methods:The mixture of Gi and GⅢ JEV isolates was inoculated on human rhabdomyosarcoma(RD).pig kidney epithelial(PS) and Aedes albopictus C6/36 clone(C6/36) which originated from human,porcine and mosquitoes,respectively.Plaque assays were performed to calculate virus titer and real-time RT-PCR with GⅠand GⅢspecific primer sets to quantify the number of GⅠ and GUI RNA copies.Results:The highest virus titer reached at the 3rd day of post infection when G Ⅰand GⅢ mixture was inoculated on RD and PS and that of C636 was at the 4^(th)day.JEVs were amplified and maintained by C6/36 cells after 10 passages whereas that by RD and PS only limited within 8 and 6 passages,respectively.GⅠ strain amplified and maintained more efficiently on C6/36 and PS but not RD.whereas GⅢ strain amplified and maintained more efficiently on RD.Conclusions:There is a correlation between the multiplication efficiency of GⅠ and GⅢ JEV strains when these two genotype strains co-infected on different cell lines with the predominance of GⅠstrains in C6/36 and PS and the limited detection of G 1 strains in RD cells proving a possible mechanism of shift JEV genotypes in nature recent decades since GⅠ emerging.

TaqMan Real-time RT-PCR Assay for Detecting and Differentiating Japanese Encephalitis Virus

Objective To detect Japanese encephalitis virus（JEV） rapidly and distinguish its genotypes, a TaqMan-based reverse transcriptase quantitative polymerase chain reaction（RT-PCR） detection system was developed.Methods By aligning the full-length sequences of JEV（G1-G5）, six sets of highly specific TaqMan real-time RT-PCR primers and probes were designed based on the highly conserved NS1, NS2, and M genes of JEV, which included one set for non-specific JEV detection and five sets for the detection of specific JEV genotypes. Twenty batches of mosquito samples were used to evaluate our quantitative PCR assay.Results With the specific assay, no other flavivirus were detected. The lower limits of detection of the system were 1 pfu/mL for JEV titers and 100 RNA copies/μL. The coefficients of variation of this real-time RT-PCR were all 〈 2.8%. The amplification efficiency of this method was between 90% and 103%.Conclusion A TaqMan real-time RT-PCR detection system was successfully established to detect and differentiate all five JEV genotypes.

Molecular Characterization of Full-length Genome of Japanese Encephalitis Virus Genotype V Isolated from Tibet, China

Objective To determine the molecular characterization of full-length genome of Japanese encephalitis virus （JEV） genotype V. Methods The full-length nucleotide sequences of JEV strains isolated from different locations and sources were used in sequence and phylogenetic analysis. Results The full-length genome of genotypes V JEV, XZ0934, and Muar strain were composed of 10 983 and 10 988 nucleotides respectively and shared a lower level of identity with JEV genotypes I-IV, ranging from 78.4% （G I, KV1899） to 79.7% （G III, JaGAr01）, for the nucleotide sequences, and from 90.0%（G I, KV1899） to 91.8%（G III, JaGAr01） for the amino acid sequences. The open reading frame （ORF） of JEV genotype V spanned nucleotides 96 to 10 397 and encoded 3 433 amino acids. Interestingly, a comparison with JEV genotype I-IV revealed that 3 nucleotides （encoded with a serine residue） were inserted in the NS4A gene of JEV genotype V, and the insertion of nucleotides was also found in downstream of the ORF stop codon in 3’-untranslated region. Moreover, numerous amino acid mutations were observed in 3 functional domains of the E gene of JEV genotype V. Conclusion The molecular characterization of JEV genotype V is significantly different from that of the known genotypes I-IV. The mutations located in the coding region and the non-coding region may be molecular markers of JEV genotype V and warrant further studies to determine their effects on biology and immunogenicity of genotype V strains.

Pharmacodynamics of aminoglycosides and tetracycline derivatives against Japanese encephalitis virus

Objective:To explore the antiviral activity of antibiotic compounds,mainly aminoglycosides and tetracyclines against Japanese encephalitis virus(JEV) induced infection in vitro.Methods:Antiviral activity were evaluated against JEV using cytopathic effect inhibition assay,virus yield reduction assay,caspase 3 level,extracellular viral detection by antigen capture ELISA and viral RNA levels.Roults:JEV induced cytopathic effect along with reduction of viral progeny plaque formation indicated antiviral potential of the compounds suggesting that antibiotics had broad spectrum activity.Doxycycline and kanamycin administration in dose dependent manner declined viral RNA replication.Conclusions:The present study shows kanamycin and doxycyclinc can affect virion structure and alter replication causing inhibition of JEV induced pathogenesis in vitro.

Seasonal abundance and potential of Japanese encephalitis virus infection in mosquitoes at the nesting colony of ardeid birds,Thailand

Objective:To investigate the abundance and seasonal dynamics of mosquitoes,and to detect Japanese encephalitis virus(JEV)in these mosquitoes at the nesting colony of ardeid birds.Methods:Mosquitoes were collected bimonthly from July 2009 to May 2010 by Centers for Disease Control.Light traps and dry ice,as a source of CO_2,were employed to attract mosquitoes.Mosquitoes were first identified,pooled into groups of upto 50 mosquitoes by species,and tested for JEV infection by viral isolation and reverse transcriptase polymerase chain reaction.Results:A total of 20370 mosquitoes comprising 14 species in five genera were collected.The five most abundant mosquito species collected were Culex tritaeniorhynchus(95.46%),Culex vishnui(2.68%),Culex gelidus(0.72%),Anopheles peditaeniatus(0.58%)and Culex quinquefasciatus(0.22%).Mosquito peak densities were observed in July.All of 416 mosquito pools were negative for JEV.Conclusions:This study provides new information about mosquito species and status of JEV infection in mosquitoes in Thailand.Further study should be done to continue a close survey for the presence of this virus in the ardeid birds.

Use of a Multiplex RT-PCR Assay for Simultaneous Detection of the North American Genotype Porcine Reproductive and Respiratory Syndrome Virus,Swine Influenza Virus and Japanese Encephalitis Virus

A multiplex reverse transcriptase-polymerase chain reaction(multiplex RT-PCR) assay was developed and subsequently evaluated for its efficacy in the detection of multiple viral infections simultaneously,in swine.Specific primers for each of the 3 RNA viruses,North American genotype porcine reproductive and respiratory syndrome virus,Japanese encephalitis virus,and swine influenza virus,were used in the testing procedure.The assay was shown to be highly sensitive because it could detect as little as 10-5 ng of each of the respective amplicons in a single sample containing a composite of all 3 viruses.The assay was also effective in detecting one or more of the same viruses in various combinations in specimens,including lymph nodes,lungs,spleens,and tonsils,collected from clinically ill pigs and in spleen specimens collected from aborted pig fetuses.The results from the multiplex RT-PCR were confirmed by virus isolation.The relative efficiency(compared to the efficiency of separate assays for each virus) and apparent sensitivity of the multiplex RT-PCR method show that this method has potential for application in routine molecular diagnostic procedures.

A Centralized Report on Pediatric Japanese Encephalitis Cases from Beijing Children's Hospital,2013

Fifteen pediatric cases of suspected Japanese encephalitis （JE） were reported in Beijing Children＇s Hospital during the late summer of 2013. The clinical manifestations in most cases included high fever, seizures, and abnormal magnetic resonance imaging findings. Twelve of 15 cases were laboratory-confirmed as JE cases by pathogen identification. Epidemiological investigations showed that five of the 12 laboratory-confirmed patients had an incomplete JE vaccination history. Follow-up investigations after discharge indicated that seven laboratory-confirmed JE patients without JE vaccinations had relatively poor prognoses, with an average Modified Rankin Scale （MRS） score of 2.6 when compared with the other five laboratory-confirmed, JE-vaccinated patients with an average MRS score of 0.5. The observation of pediatric JE cases among those with a history of JE vaccination warrants further attention.

The Relationship between Japanese Encephalitis and Environmental Factors in China Explored Using National Surveillance Data

Japanese encephalitis（JE）is a serious public health issue.This study was undertaken to better understand the relationship between JE distribution and environmental factors in China.JE data from 2005 to 2010 were retrieved from National Notifiable Disease Report System.

Epidemiological Characteristics of Japanese Encephalitis in Guizhou Province,China,1971-2009

Objective The aim of the study was to establish the contemporary epidemiological characteristics of Japanese encephalitis （JE） in Guizhou Province. Methods A retrospective study of National Notifiable Disease Reporting System （NNDRS） data from 2971 through 2009, was conducted to ascertain the geographical, seasonal, and age distributions of JE incidence in Guizhou Province, China. Results A total of 68 425 JE cases were reported in Guizhou from 1971-2009. The JE cases occurred sporadically in all 9 prefectures of Guizhou, mostly among residents of rural areas. Seasonal distribution of JE remained consistent over the period from 1971-2009 with the main transmission season starting from June to September and peaking in August. JE occurred mainly in children under the age of 15 years with peak incidence in the 0-6-year age group. Pearson＇s correlation analysis showed that JE vaccine distribution had a negative correlation with JE incidence rates during 1971-2009 （coefficient of correlation=-0.475, P〈O.01）. Conclusion Over the period of 1971-2009, the JE incidence rate had declined dramatically in terms of geographical and age distributions due to JE vaccination to children at risk.

Japanese Encephalitis in China in the Period of 1950–2018:From Discovery to Control

Japanese encephalitis(JE)was first discovered in Japan in 1871;in 1924,a major outbreak occurred,with 6,000 JE cases reported and a mortality rate of approximately 60%[1,2].Later studies showed that JE is caused by the Japanese encephalitis virus(JEV),which is spread by mosquitoes.

Japanese encephalitis following liver transplantation: A rare case report

BACKGROUND Japanese encephalitis(JE) is a serious public health concern with a high mortality rate in many Asian countries. For many years, JE virus(JEV) was considered the major cause of viral encephalitis in Asia. Although most JE cases are asymptomatic, the case fatality rate approaches 30%, and approximately 30%–50% of survivors have long-term neurological sequelae. To the best of our knowledge, JEV infection has never been reported following liver transplantation.CASE SUMMARY We report a case of a woman who underwent liver transplantation for autoimmune liver disease but presented with fever and neurological symptoms 13 d after transplantation. Magnetic resonance imaging revealed JEV infection,and positive immunoglobulin M antibody to JEV in blood and cerebrospinal fluid confirmed JE. The patient was treated with antiviral agents, immune regulation,and organ function support. No neurological sequelae were present after 1 year of follow-up.CONCLUSION Imaging and lumbar puncture examination should be performed as soon as possible in patients with fever and central nervous system symptoms after liver transplantation, and the possibility of atypical infection should be considered,which is helpful for early diagnosis and improved prognosis.

Influence of socio-economic status and environmental factors on serologically diagnosed Japanese encephalitis cases in the state of West Bengal, India during 2005-2010

Objectives: The main aim of the current study is to examine the influence of socio-economic status and environmental factors on serologically diagnosed Japanese encephalitis cases in the state of West Bengal, India during 2005-2010. Materials and methods: A total of 648 blood/CSF specimens were collected and/or referred from the suspected AES cases, admitted in the different medical colleges and hospitals of the state during the year of 2005-2010. These specimens were subjected to JE Mac ELISA to determine the actual JE case amongst these AES. The association of the socio-economic status and environmental factors with the serologically diagnosed JE positive cases was studied by a statistical analysis through Normal Deviate test or Z test. Result: Out of 648 specimens, only 175 (27.0%) specimens were reactive to JE IgM antibody, of which 60.0% were from the male individuals and 40.0% from the female population. Major cases were observed in the age group of 0 - 10 years;followed by 11 - 20 years. Regarding literacy, only 58.3% cases had no education and 41.7% were from the literate with varying level of education, i.e., from primary level to post gra- duate level. A total of 65.7% cases were from low income group where as only 34.3% cases were from high income group. Regarding house type, 62.3% cases lived in mud house and 37.7% cases lived in the brick house. In most of the cases (74.3%), persons were living in close proximity to rice fields/lakes/ponds. 69.7% cases were found to occur in the monsoon and post-monsoon period whereas 30.3% cases were reported in the pre-monsoon period. Conclusion: Our study concludes that socio-economic status and environmental conditions were statistically significant contextual risk factors for serologically diagnosed JE incidences in West Bengal where JE is proved to be endemic in nature and such study constitutes a new report of this kind in the region.

Development of an Indirect ELISA Using Recombinant Truncated Envelope Glycoprotein for Detection of Antibodies against Japanese Encephalitis Virus

[ Objective] To develop an indirect ELISA assay for detecting antibodies against envelope glycoprotein （ E protein） of Japanese encephalitis virus （JEV）. [ Method] Specific primers were designed according to JEV sequences published in the GenBank. The cDNA of JEV E gene （about 1 000 10p） was amplified by the RT-PCR with the specific primers. After sequencing analysis, the E gene was cloned into pET30a expression vector and expressed in E. coli BL21 （DE3） with the induction of IPTG. After denaturation, purification and renaturation, the recombinant protein was analyzed by the SDS-PAGE and the westem blotting. An indirect ELISA was developed to detect antibodies against JEV. [ Result] The E protein was mainly expressed in inclusion body. With the purified E protein, the indirect ELISA was developed and displayed good specificity, sensitivity and repeatability, [ Conclusion]The developed ELISA using the truncated E protein as antigen is a simple, convenient and rapid serological method for diagnosis, monitoring antibody level and epidemiological investigation of JEV.

Time Series Models for Short Term Prediction of the Incidence of Japanese Encephalitis in Xianyang City, P R China

为在 Xianyang 预报日本脑炎(JE ) 的流行病构造季节的 Autoregressive 综合动人的一般水准(SARIMA ) 的一个模型的目的， Shaanxi，中国，并且为 JE 控制和 prevention.Methods 提供珍贵引用信息理论上流行病的学习在研究进程被采用。为到 2014 年 9 月的从 2005 年 1 月的时期的 JE 上的每月的发生数据在 Xianyang 在疾病预防和控制的中心从一个被动监视系统被获得， Shaanxi 省。一个最佳的 SARIMA 模型与盒子和 Jenkins 途径从 2005 ～ 2013 为 JE 发生被开发。这个 SARIMA 模型能为一年 2014 和 2015 .Results SARIMA 预言 JE 发生( 1 ， 1 ， 1 )( 2 ， 1 ， 1 )12被认为是有最低贝叶斯的信息标准的最好的模型， Akaike 信息标准，吝啬的绝对错误价值，最高的 R 2,和一个更低的吝啬的绝对百分比错误。SARIMA (1， 1， 1 )(2， 1， 1 )12 为在 Xianyang 预言 JE 发生静止、精确。预言的发生，在 0.3/100 附近 000 从 6 月到有低错误的在 2014 的 8 月，与实际发生相比是更高的。因此， SARIMA (1， 1， 1 )(2， 1， 1 )12 看起来可靠、精确并且能被用于建议预言模型能提供的发生 prediction.Conclusions 反常地被增加的 JE 发生的早鉴定的线索(0.4/100 000 ) 。根据在 2014 的预言的结果，在 Xianyang 的 JE 发生将稍微衰退并且从 6 月到达它的山峰到 8 月。

Preparation of Polyclonal Antibodies against NS3 Protein of Japanese Encephalitis Virus

Japanese encephalitis （JE） is a central nervous system disease caused by Japanese encephalitis virus （JEV）, which can infect human and a variety of animals and cause irreversible nerve damages. NS3 protein plays an important role in the process of JEV polyprotein hydrolysis, which is essential for JEV replication. Therefore, NS3 protein may be used as a potential drug target to treat Japanese encephalitis. In this study, the pET-28a-NS3 plasmid was successfully constructed and expressed in E. coli BL21 （ DE3 ） under IPTG induction. The molecular weight of the expressed recombinant protein was 55 ku, which was consistent with the expected result. The positive serum was prepared by immunizing BALB/c mice with NS3 protein and identified by indirect immunofluorescence （IFA）. The results showed that there was a fluorescence reaction between the prepared positive serum of NS3 protein and cells infected with JEV.

Inhibition of Japanese Encephalitis Virus Infection by Flavivirus Recombinant E Protein Domain Ⅲ

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus closely related to the human pathogens including yellow fever virus, dengue virus and West Nile virus. There are currently no effective antiviral therapies for all of the flavivirus and only a few highly effective vaccines are licensed for human use. In this paper, the E protein domain III (DIII) of six heterologous flaviviruses (DENV1-4, WNV and JEV) was expressed in Escherichia coli successfully. The proteins were purified after a solubilization and refolding procedure, characterized by SDS-PAGE and Western blotting. Competitive inhibition showed that all recombinant flavivirus DIII proteins blocked the entry of JEV into BHK-21 cells. Further studies indicated that antibodies induced by the soluble recombinant flavivirus DIII partially protected mice against lethal JEV challenge. These results demonstrated that recombinant flavivirus DIII proteins could inhibit JEV infection competitively, and immunization with proper folding flavivirus DIII induced cross-protection against JEV infection in mice, implying a possible role of DIII for the cross-protection among flavivirus as well as its use in antigens for immunization in animal models.