Serological Investigation into the Infected Genotypes of Patients with Japanese Encephalitis in the Coastal Provinces of China

Objective Genotypes(G)1,3,and 5 of the Japanese encephalitis virus(JEV)have been isolated in China,but the dominant genotype circulating in Chinese coastal areas remains unknown.We searched for G5 JEV-infected cases and attempted to elucidate which JEV genotype was most closely related to human Japanese encephalitis(JE)in the coastal provinces of China.Methods In this study,we collected serum specimens from patients with JE in three coastal provinces of China(Guangdong,Zhejiang,and Shandong)from 2018 to 2020 and conducted JEV cross-neutralization tests against G1,G3,and G5.Results Acute serum specimens from clinically reported JE cases were obtained for laboratory confirmation from hospitals in Shandong(92 patients),Zhejiang(192 patients),and Guangdong(77 patients),China,from 2018 to 2020.Seventy of the 361 serum specimens were laboratory-confirmed to be infected with JEV.Two cases were confirmed to be infected with G1 JEV,32 with G3 JEV,and two with G5 JEV.Conclusion G3 was the primary infection genotype among JE cases with a definite infection genotype,and the infection caused by G5 JEV was confirmed serologically in China.

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.

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.

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.

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.

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.

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.

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

Objective To construct a model of Seasonal Autoregressive Integrated Moving Average （SARIMA） for forecasting the epidemic of Japanese encephalitis （JE） in Xianyang, Shaanxi, China, and provide valuable reference information for JE control and prevention. Methods Theoretically epidemiologic study was employed in the research process. Monthly incidence data on JE for the period from Jan 2005 to Sep 2014 were obtained from a passive surveillance system at the Center for Diseases Prevention and Control in Xianyang, Shaanxi province. An optimal SARIMA model was developed for JE incidence from 2005 to 2013 with the Box and Jenkins approach. This SARIMA model could predict JE incidence for the year 2014 and 2015. Results SARIMA （1, 1, 1） （2, 1, 1）12 was considered to be the best model with the lowest Bayesian information criterion, Akaike information criterion, Mean Absolute Error values, the highest R2, and a lower Mean Absolute Percent Error. SARIMA （1, 1, 1） （2, 1, 1）12 was stationary and accurate for predicting JE incidence in Xianyang. The predicted incidence, around 0.3/100 000 from June to August in 2014 with low errors, was higher compared with the actual incidence. Therefore, SARIMA （1, 1, 1） （2, 1, 1）12 appeared to be reliable and accurate and could be applied to incidence prediction. Conclusions The proposed prediction model could provide clues to early identification of the JE incidence that is increased abnormally （≥0.4/100 000）. According to the predicted results in 2014, the JE incidence in Xianyang will decline slightly and reach its peak from June to August.The authors wish to thank the staff from the CDCs from 13 counties of Xianyang, Shaanxi province, China, for their contribution to Japanese encephalitis cases reporting.

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.

Surface Display of Domain Ⅲ of Japanese Encephalitis Virus E Protein on Salmonella Typhimurium by Using an Ice Nucleation Protein

A bacterial cell surface display technique based on an ice nucleation protein has been employed for the development of live vaccine against viral infection. Due to its ubiquitous ability to invade host cells, Salmonella typhimurium might be a good candidate for displaying viral antigens. We demonstrated the surface display of domain III of Japanese encephalitis virus E protein and the enhanced green fluorescent protein on S. typhimurium BRD509 using the ice nucleation protein. The effects of the motif in the ice nucleation protein on the effective display of integral protein were also investigated. The results showed that display motifs in the protein can target integral foreign protein on the surface of S. typhimurium BRD509. Moreover, recombinant strains with surface displayed viral proteins retained their invasiveness, suggesting that the recombinant S. typhimurium can be used as live vaccine vector for eliciting complete immunogenicity. The data may yield better understanding of the mechanism by which ice nucleation protein displays foreign proteins in the Salmonella strain.

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 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.

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.

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.

Study on DNA Immunization by Recombinants Encoding Japanese Encephalitis Virus prME and E Proteins

To study the expression characteristic of Japanese encephalitis virus (JEV) prME and E proteins and the efficacy of DNA immunization by different recombinant plasmids containing JEV prME (2001 bp) and E (1500 bp) genes, two recombinants (pJME and pJE) containing JEV prME and E genes fused with FLAG were constructed and then transfected into HepG2 and COS-1 cells by liposome fusion. The expression feature of FLAG-prME (about 72 kDa) and FLAG-E (about 54 kDa) proteins in transfected cells were analyzed by Western blot and two antibody systems (anti-FLAG and anti-E). BALB/c mice were immunized with 100 μg of two kinds of recombinants by intramuscular injection, and JEV JaGAr-01 strains (10 5 PFU/100 μl)were given to BALB/c mice by intraperioneal injection 3 wk after twice DNA immunization by a lethal virus challenge. BALB/c mice were observed for 21 days after challenge. 80% plaque reduction neutralization test was performed to titrate neutralization antibody before and after viral challenge. It was found that the expression of proteins associated with pJME and pJE was determined in transfected cells with anti-FLAG and a new protein of 11 kDa was detected in HepG2 and COS-1 cells transfected with pJME. Only E (53 kDa) protein was identified as transfected with pJME using anti-E. Higher level of neutralization antibodies and the efficacy of protective immunity were induced with pJME immunization, and were similar to those induced by inactivated Japanese encephalitis vaccine, but were better than those induced with pJE. It concludes that the expression level from prM to E proteins of JEV is different in vitro, and the in vitro expression efficiency of pJME was better than that of pJE. FLAG-prME protein expressed by pJME could be cleaved by peptidase from host. The efficacy of DNA immunization is correlated to the expression characterization of related proteins expressed in vitro.

Prevalence and risk factors associated with Japanese encephalitis virus infection in swine population of Assam,India

Objective:To assess the prevalence of Japanese encephalitis virus(JEV)and associated risk factors in the swine population of Assam.Methods:A total of 432 swine serum and blood samples were collected from Barpeta and Sonitpur districts of Assam and were screened for the presence of JEV antibodies.Information related to risk factors was collected using a self-designed questionnaire from 120 swine-rearing farmers.Linear-mixed models were used for prevalence estimation.Univariate and multivariate regression models were constructed to evaluate the association of demography,season and management practices with JEV positive status.Results:Overall,the JEV infection prevalence was 51.6%at farm and 47.1%at slaughter premises.Phylogenetic analysis of partial sequence of envelope gene of two positive field samples revealed that both isolates belonged to genotypeⅢJEV.Isolate 1 shared a common clade with human isolates while isolate 2 belonged to the same clade as that of other JEV swine strain isolated from India.The final multivariate model showed that two factors including monsoon season(Adjusted OR 5.6;95%CI 2.1-14.9;P<0.001)and water logging in the area near the pig shelter(Adjusted OR 16.9;95%CI 6.1-47.3;P<0.001)were associated with greater odds of swine being infected with JEV.Conclusions:High prevalence of JEV in swine population of Assam state indicates a significant risk of virus transmission to humans while risk factor study underlines the urgent need for awareness campaigns in the Assam.

E Sequence Analysis of Persistently Infected Mutant Japanese Encephalitis Virus Strains

Summary： A persistent infection model was established after human hepatoma cells infected by Japanese encephalitis viruses were subcultured for several times. Viral titers of mutant viruses in persistently infected cells were examined by plaque methods using BHK cells. Nucleotides of the E coding region of two wild and two mutant viruses were amplified by RT-PCR. PCR products were sequenced by ABI-PRSM^TM310 sequencing system. Compared to JaGAr-01 wild strains, four amino acids were replaced （E61Tyr→Asp, E219His→Tyr, E384Val→Glu, E418Pro→Ala） in the E sequence of JaGAr-01 persistently-infected mutant strains. Eleven amino acid replacement （E51Arg →Ser, E61Tyr→Asp, E83Lys→Glu, E123Ser→Arg, E209Arg→Lys, E227Pro→Ser, E276Asp→er, E290Arg→Lys, E387Lys→Arg, E418Leu→Pro, E454Arg→Gly） was also noted when we compared the E sequence between persistently infected Nakayama and its wild strains. A lot of similarities of amino acid sequence between mutant strains JaGAr-01 and Nakayama were also noted. It was concluded that geno-variation existed in E region of mutant viruses and the mutant protein encoded by E region, especially the mutation of E61 （Tyr→Asp） may contribute to the maintenance of the persistent infection of Japanese encephalitis virus.

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.

Phylogeny and Homologous Recombination in Japanese Encephalitis Viruses

Japanese encephalitis virus（JEV） is a significant causative agent of arthropod-borne encephalitis and what is less clear that the factors cause the virus wide spread. The objective was to confirm whether the homologous recombination imposed on JEV. The phylogenetic and homologous recombination analyses were performed based on 163 complete JEV genomes which were recently isolated. They were still separated into five genotypes（GI-GV） and the most of recently isolated JEVs were GI rather than GIII in Asian areas including China's Mainland. Two recombinant events were identified in JEV and the evidence of the recombination was observed between China and Japan isolates that partitioned into two distinct subclades, but still the same genotype（GIII）. Our data further suggested that most of the nucleotides in JEV genome were under negative selection; however, changes within codon 2 316（amino acid NS4b-44） showed an evidence of the positive selection.

Cloning and Sequence Analysis of the Full-Length Genome of Japanese encephalitis virus Strain SXBJ07 Isolated from Swine

A virus strain, showing cytopathic effect in BHK-21 cell, was isolated from swine brains in Shaanxi Province, China, in 2007. The isolate was confirmed as Japanese encephalitis virus （JEV） by immunofluorescence assay （IFA） and reverse transcription polymerase chain reaction （RT-PCR）, and named SXBJ07. The complete nueleotide and deduced amino acid sequences of the JEV strain SXBJ07 were determined. Its single open reading frame has a total of 3 432 amino acid residues. An extensive E gene based phylogenetic analysis was performed, the result showed that SXBJ07 strain belongs to genotype I. Comparison of the SXBJ07 genomic sequence with those of the 24 fully sequenced JEV strains in published databases showed nucleotide homology ranging from 99.0 to 83.7%; amino acid homology ranged from 99.8 to 94.8%. Compared SXBJ07 with SA14-14-2 strain, the current live vaccine strain in China, the homology of amino acid in envelope gene was 97.0%; and there were amino acid substitutions in 13 sites of the active domains of E protein （E1-E411）.