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.

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.

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.

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.

Construction and Preliminary Application of RT-LAMP Detection Method for Swine Japanese B Encephalitis Virus

[ Objective] This study aimed to establish a rapid, sensitive and specific method using reverse transcription loop-mediated isothermal amplification （RT-LAMP） technology to detect swine Japanese B encephalitis virus （JEV）. [ Method ] Four specific LAMP primers were designed according to six loci the conservative region of JEV E gene sequence. Positive JEV RNA sample was used as a template for one-step amplification, and the reaction conditions and reaction system were optimized. [ Result] Experimental results showed that the established method had high sensitivity, with the detection limit of 0.5pg; specificity experi- ments indicated that the method had high specificity and there was no amplification reaction for other viral pathogens. The coincidence rate between detection results of RT-LAMP and RT-Nested-PCR was 90.9%. After RT-LAMP reaction, a chemiluminescent agent was added for visual observation, which greatly reduced the detection time. This method required no special equipment but only a water bath, which was a simple, sensitive and rapid detection method for swine Japanese B encephalitis virus and could be applied in primary laboratories. [ Conclusion] An RT-LAMP detection method for swine Japanese B encephalitis virus was successfully established and preliminarily applied in clinical practice.

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）.

Japanese Encephalitis Virus Replication and Inhibitory Effect of shRNA in Mice

Japanese Encephalitis Virus (JEV) is responsible for over 30,000 annual cases of encephalitis worldwide, causing 30% mortality. JEV is thus a continuing threat to public health, so development of new antiviral drugs against JEV is desirable. Here, we examined JEV replication in mouse and used a short hairpin RNA JRi as the antiviral agent. The features of virus replication in neuron and survival rates of mice infected with JEV were different between virus strains. The mice infected with the virulent JEV strain (JaGAr01) were injected with pJRi (100 μg/mouse) which produced shRNAJRi. The survival rates of mice treated at 3 days before, the same day and 3 days after JEV infection were 22%, 78% and 44%, respectively. In addition, we demonstrated that the injection of pJRi induced interferon (IFN) production in cells and mice. These results suggest that the replication of JEV can be efficiently inhibited by RNAi and innate immunity including IFN. These data mean that pJRi has the inhibitory activity against JEV infection in vivo, and could be used as an antiviral drug to treat JEV infection.

Herpes Simplex Virus Encephalitis Complicated by Acute Ischemic Stroke

Introduction: Herpes simplex virus is the most common etiology for life-threatening sporadic encephalitis worldwide. Antiviral therapy with acyclovir has been shown to reduce mortality and should be started promptly in patients with clinically suspected viral encephalitis before serological confirmation of the diagnosis. Despite antiviral treatment, it is associated with significant mortality and a wide range of neurologic sequelae or neuropsychiatric disorders. Clinical presentation includes fever, headache, altered mental status, and focal or generalized seizures. In some cases, it can present with focal neurological deficits, such as an acute stroke. The aim of this study is to identify rare complications of HSVE. Presentation: We present a case of a 71-year-old female patient with herpes virus encephalitis and an ischemic cerebral accident. The findings of CT scan of the brain revealed an extensive right temporal hypodensity. CSF findings include an elevated protein level, normal glucose level and pleocytosis with lymphocyte predominance. The lumbar tap confirmed the presence of herpes simplex virus type 1 with polymerase chain reaction (PCR) in the CSF. Neurological manifestations include focal neurological deficit with left-sided hemiparesis and coma. After 40 days of complex therapy, an improvement in the mental state was observed. Conclusion: There are varying degrees of neurologic sequelae among survivors in children and adults despite the antiviral treatment. Herpes simplex encephalitis has significant morbidity and high mortality due to the lack of prophylactic treatment and preventable strategies.

Genetically engineered bacterial-like particles induced specific cellular and humoral immunity as effective tick-borne encephalitis virus vaccine

Tick-borne encephalitis(TBE)is a natural focal disease with fatal encephalitis induced by tick-borne encephalitis virus(TBEV),seriously threatening human and public health.Protection of TBE depends on vaccination with inactivated vaccine,which requires high cost and multiple immunizations.Here,we construct genetically engineered bacterial-like particles(BLPs)as an effective TBEV vaccine with simplified immunizations and improved immune efficacy.The TBEV BLPs involve the combination of the gram-positive enhancer matrix from Lactococcus lactis,and TBEV envelope(E)protein expressed by genetically engineered recombinant baculovirus.The prepared TBEV BLPs can effectively stimulate the activation of dendritic cells to present the TBEV E proteins to T and B cells,leading to strong and durable cellular and humoral immune responses in mice.Surprisingly,the serum levels of specific IgG antibodies in mice remain about 10^(6)at 6 months after the secondary immunization.Overall,the TBEV BLPs can be used as a potent vaccine candidate,laying the foundation for developing novel TBEV genetically engineered vaccines.

Development of a novel virus-like particle-based vaccine for preventing tick-borne encephalitis virus infection

Tick-borne encephalitis virus(TBEV)is an important tick-borne pathogen that poses as a serious public health concern.The coverage and immunogenicity of the currently available vaccines against TBEV are relatively low;therefore,it is crucial to develop novel and effective vaccines against TBEV.The present study describes a novel strategy for the assembly of virus-like particles(VLPs)by co-expressing the structural(core/prM/E)and non-structural(NS2B/NS3Pro)proteins of TBEV.The efficacy of the VLPs was subsequently evaluated in C57BL/6 mice,and the resultant IgG serum could neutralize both Far-Eastern and European subtypes of TBEV.These findings indicated that the VLP-based vaccine elicited the production of cross-subtype reactive antibodies.The VLPs provided protection to mice lacking the type I interferon receptor(IFNAR^(-/-))against lethal TBEV challenge,with undetectable viral load in brain and intestinal tissues.Furthermore,the group that received the VLP vaccine did not exhibit significant pathological changes and the inflammatory factors were significantly suppressed compared to the control group.Immunization with the VLP vaccine induced the production of multiple-cytokine-producing antiviral CD4+T cells in vivo,including TNF-α^(+),IL-2^(+),and IFN-γ^(+)T cells.Altogether,the findings suggest that noninfectious VLPs can serve as a potentially safe and effective vaccine candidate against diverse subtypes of TBEV.

Isolated Hyperacute T-Waves in West Nile Encephalitis Indicating Atypical Variant of Stress-Induced Cardiomyopathy

Several cardiac outcomes have been reported with West Nile-encephalitis;however, the underlying pathophysiology remains complex. We present a 42-year-old female, with multiple sclerosis, whose neurological symptoms and respiratory decline were finally explained by the diagnosis of West Nile-encephalitis. During her admission, the isolated peaked T-waves indicated the underlying stress-induced cardiomyopathy. The absence of all other causes of hyperacute T-waves, their subsequent resolution with the resolution of infection and improvement in wall motion abnormalities, further supported the association. This case highlights the importance of considering hyperacute T-waves in an approach towards the diagnosis of WNV-encephalitis related atypical variant of stress-induced cardiomyopathy.

Pigsties near dwellings as a potential risk factor for the prevalence of Japanese encephalitis virus in adult in Shanxi,China

Background:The increasing trend of adult cases of Japanese encephalitis(JE)in China,particularly in northern China,has become an important public health issue.We conducted an epidemiological investigation in the south of Shanxi Province to examine the relationships between mosquitoes,Japanese encephalitis virus(JEV),and adult JE cases.Methods:Mosquito specimens were collected from the courtyards of farmers’households and pig farms in Shanxi Province.Mosquitoes were pooled,homogenized,and centrifuged.Reverse transcription-polymerase chain reaction(RT-PCR)was used to detect mosquito-borne arbovirus genes in homogenates.Specimens positive for these genes were inoculated into the baby hamster kidney cell line(BHK-21)to isolate virus.Minimum infection rate was calculated and phylogenetic analyses were performed.Results:A total of 7943 mosquitoes belonging to six species in four genera were collected;Culex tritaeniorhynchus accounted for 73.08%(5805/7943),C.pipiens pallens for 24.75%(1966/7943),and the remaining 3%(104/7943)consisted of Anopheles sinensis,Aedes vexans,Ae.dorsalis,and Armigeres subalbatus.Sixteen pools were positive for JEV based on RT-PCR using JEV pre-membrane gene nested primers.Phylogenetic analyses showed that all JEVs belonged to genotype I;two pools were positive using Getah Virus(GETV)gene primers.In addition,one JEV strain(SXYC1523)was isolated from C.pipiens pallens specimens.These results indicate that the minimum infection rate of JEV in mosquito specimens collected from the courtyards of farmers’households with pigsties was 7.39/1000;the rate for pig farms was 2.68/1000;and the rate for farmers’courtyards without pigsties was zero.Conclusions:The high-prevalence regions of adult JE investigated in this study are still the natural epidemic focus of JEV.Having pigsties near dwellings is a potential risk factor contributing to the prevalence of adult JE.To prevent the occurrence of local adult JE cases,a recommendation was raised that,besides continuing to implement the Expanded Program on Immunization for children,the government should urge local farmers to cease raising pigs in their own courtyards to reduce the probability of infection with JEV.

PD1^+CCR2^+CD8^+T Cells Infiltrate the Central Nervous System during Acute Japanese Encephalitis Virus Infection

Japanese encephalitis(JE)is a viral encephalitis disease caused by Japanese encephalitis virus(JEV)infection.Uncontrolled inflammatory responses in the central nervous system(CNS)are a hallmark of severe JE.Although the CCR2-CCL2 axis is important for monocytes trafficking during JEV infection,little is known about its role in CNS trafficking of CD8^+T cells.Here,we characterized a mouse model of JEV infection,induced via intravenous injection(i.v.)and delineated the chemokines and infiltrating peripheral immune cells in the brains of infected mice.The CNS expression of chemokines,Ccl2,Ccl3,and Ccl5,and their receptors,Ccr2 or Ccr5.was significantly up-regulated after JEV infection and was associated with the degree of JE pathogenesis.Moreover,JEV infection resulted in the migration of a large number of CD8^+T cells into the CNS.In the brains of JEV-infected mice,infiltrating CD8^+T cells expressed CCR2 and CCR5 and were found to comprise mainly effector T cells(CD44^+CD62L_).JEV infection dramatically enhanced the expression of programmed death 1(PD-1)on infiltrating CD8^+T cells in the brain,as compared to that on peripheral CD8^+T cells in the spleen.This effect was more pronounced on infiltrating CCR2^+CD8^+T cells than on CCR2-CD8^+T cells.In conclusion,we identified a new subset of CD8^+T cells(PD1^+CCR2^+CD8^+T cells)present in the CNS of mice during acute JEV infection.These CD8^+T cells might play a role in JE pathogenesis.

Recovery of a Far-Eastern Strain of Tick-Borne Encephalitis Virus with a Full-Length Infectious cDNA Clone

Tick-borne encephalitis virus(TBEV) is a pathogenic virus known to cause central nervous system(CNS) diseases in humans, and has become an increasing public health threat nowadays. The rates of TBEV infection in the endemic countries are increasing. However, there is no effective antiviral against the disease. This underscores the urgent need for tools to study the emergence and pathogenesis of TBEV and to accelerate the development of vaccines and antivirals. In this study, we reported an infectious c DNA clone of TBEV that was isolated in China(the WH2012 strain). A beta-globin intron was inserted in the coding region of nonstructural protein 1(NS1) gene to improve the stability of viral genome in bacteria. In mammalian cells, the inserted intron was excised and spliced precisely, which did not lead to the generation of inserted mutants. High titers of infectious progeny viruses were generated after the transfection of the infectious clone. The cDNA-derived TBEV replicated efficiently, and caused typical cytopathic effect(CPE) and plaques in BHK-21 cells. In addition, the CPE and growth curve of cDNA-derived virus were similar to that of its parental isolate in cells. Together, we have constructed the first infectious TBEV cDNA clone in China, and the clone can be used to investigate the genetic determinants of TBEV virulence and disease pathogenesis, and to develop countermeasures against the virus.

DC-SIGN promotes Japanese encephalitis virus transmission from dendritic cells to T cells via virological synapses

Skin-resident dendritic cells(DCs) likely encounter incoming viruses in the first place, and their migration to lymph nodes following virus capture may promote viral replication. However, the molecular mechanisms underlying these processes remain unclear. In the present study, we found that compared to cell-free viruses, DC-bound viruses showed enhanced capture of JEV by T cells.Additionally, JEV infection was increased by co-culturing DCs and T cells. Blocking the C-type lectin receptor DC-specific intercellular adhesion molecule-3-grabbing non-integrin(DC-SIGN) with neutralizing antibodies or antagonists blocked JEV transmission to T cells. Live-cell imaging revealed that DCs captured and transferred JEV viral particles to T cells via virological synapses formed at DC-T cell junctions. These findings indicate that DC-SIGN plays an important role in JEV transmission from DCs to T cells and provide insight into how JEV exploits the migratory and antigen-presenting capabilities of DCs to gain access to lymph nodes for dissemination and persistence in the host.

Tumor suppressor p53 functions as an essential antiviral molecule against Japanese encephalitis virus

Japanese encephalitis virus （jEV） is a mosquito-borne virus of the family Flaviviridae. It is the causative agent of Japanese encephalitis with approximately 50,000 infection cases and 10,000 fatal cases annually in Asia （Erlanger et al., 2009）. Although liveattenuated JEV vaccine has been developed and used for human and pig vaccination, JE occurs epidemically or sporadically in some developing countries or even in vaccinated areas （Solomon, 2006）. Host resistance factors play an important role in the outcome of viral infection.