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 S...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.展开更多
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 homologou...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.展开更多
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 a...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.展开更多
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 mechan...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.展开更多
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 evaluate...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.展开更多
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 bimo...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.展开更多
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...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.展开更多
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...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.展开更多
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.Speci...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.展开更多
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 therap...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.展开更多
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, Salmonel...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.展开更多
[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 sequence...[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 bp) 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 western 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.展开更多
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 importa...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.展开更多
Objective:To report high co-positivity of anti-dengue virus(DV)and anti-Japanese encephalitis virus(JEV)IgM in an area endemic for both the viruses and to discuss the possibilities of coinfection.Methods:Serum samples...Objective:To report high co-positivity of anti-dengue virus(DV)and anti-Japanese encephalitis virus(JEV)IgM in an area endemic for both the viruses and to discuss the possibilities of coinfection.Methods:Serum samples from the patients who presented with fever,suspected central nervous system infection and thrombocytopenia,were tested for anti-DV IgM and antiJEV IgM antibodies.Conventional reverse transcriptase polymerase chain reaction was done for detection of DV RNA and JEV RNA.Results:Of 1 410 patient sera tested for anti-DV and antiJEV antibodies,129(9.14%)were co-positive for both.This co-positivity was observed only in those months when anli-JEV IgM positivily was high.Tilers of both anli-DV IgM and anti-JEV IgM were high in most of the co-positive eases.Among these 129 co-positive cases,76 were lesled by conventional reverse Iranscriplase polymerase chain reaction for both flaviviruses,of which eight cases were co-positive for DV and JEV.Conclusions:Co-infection with more than one fluvivirus species can occur in hyperendemic areas.展开更多
Objective:To assess the virus infection in mosquitoes during different seasons and correlated with various climatic factors.Methods:The field collected vectors were screened for Japanese encephalitis(JE) virus after d...Objective:To assess the virus infection in mosquitoes during different seasons and correlated with various climatic factors.Methods:The field collected vectors were screened for Japanese encephalitis(JE) virus after dessication using ELISA method.Most of the positive pools were recorded from Culex tritaeniorhynchus(Cx.tritaeniorhynchus) and Culex.gelidus(Cx.gelidus) during JE transmission season(winter) and some positive pools were also reported during non JE transmission periods(i.e.summer and rainy seasons).Results:The minimum infection rates(MIR) of 1.75 from Cx.tritaeniorhynchus and 0.17 from Cx.gelidus has been reported in the year 2002 at the beginning of the study and the values were found nil at the end of the study(2006) from the study areas of Kurnool district.Conclusions:From this study it is noted that MTR of Cx.gelidus and Cx.tritaeniorhynchus were modulated by various meteorological parameters.The mosquito vector abundance increases after the monsoon period(winter) and lowest in dry season(summer). Similarly,MIR fluctuated between seasons with higher MIR recorded after monsoon period and lower in the rest of season.Impact of these metrological parameters in JE virus infected mosquitoes is discussed in this paper.展开更多
To investigate the potential utility of nuclease--resistant oligodeoxynucleotides (S-ODN) as anew class of antiviral agents. Methods: Two antisense phosphorothioate analogues (20--iner) complementary to thesequences o...To investigate the potential utility of nuclease--resistant oligodeoxynucleotides (S-ODN) as anew class of antiviral agents. Methods: Two antisense phosphorothioate analogues (20--iner) complementary to thesequences of the first AUG codon and 5’ terminus of NSS of JEV SA--14 genome have been synthesized and their effects on CPE, viral antigen expression and virus plaque formation were tested in vitro. Results: The resultsshowed that 1. 0 pmol/L of S-ODN greatly deferred the onset of CPE in cultured BHKZI cells for at least 48 h.Addition of 5. 0 pmol/L or more S--ODN to culture medium fluid, 2 h prior to 100TCID,,virus inoculum, notablysuppressed viral antigen expression in the cells by making it lower than the limit of EIA detection in 48 h. The inhibition lasted more than 96 h. Viral plaque assay results demonstrated that S-ODN were most effect’ive within 18h with plaque inhibition rate over 90% by 5. 0 pmol/L S--ODNI. The inhibitory activity soon faded in 24 h. In addition, high concentrations (up to 80. 0 pmol/L) of S--ODN did not show any obvious cytotoxicity in 6 d by usingtrypan blue dye exclusion method. Conclusion: The specific synthetic S--ODN transitorily inhibited JEV replicationin BHK--ZI cells with characteristics of specificity and S--ODN dose--dependence.展开更多
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) gene...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.展开更多
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) a...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 nucleotide 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).展开更多
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 per- sistently infected cells wer...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 per- sistently 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-PRSMTM310 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→Ser, 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 con- cluded 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 persis- tent infection of Japanese encephalitis virus.展开更多
A low-density oligonucleotide microarray was used for the detection of Japanese encephalitis virus (JEV) , combining with restriction display PCR labeling method. The hybridization targets were amplified from 6 plasmi...A low-density oligonucleotide microarray was used for the detection of Japanese encephalitis virus (JEV) , combining with restriction display PCR labeling method. The hybridization targets were amplified from 6 plasmids containing several JEV gene fragments. Corresponding oligonucleotide probe spots were detected unambiguously. We claim that the oligonucleotide microarray technology is feasible and may have potential for clinical laboratory application.展开更多
基金financially supported by the Department of Biotechnology,Government of India,through the research project entitled‘Genomics assisted pathobiology to identify novel targets for diagnosis and therapeutic intervention(s)of Japanese encephalitis and Leptospirosis’。
文摘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.
基金Supported by the National Natural Science Foundation of China(31272569,31270187)Projects in the National Science&Technology Pillar Program during 12th Five-year Plan Period(2013BAD12B04)Harbin Science and Technology Bureau(RC2012XK002003)
文摘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.
文摘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.
基金funded by Vietnam National Foundation for ScienceTechnology Development(NAFOSTED)under grant number:106.16-2011.68
文摘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.
文摘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.
基金supported by the Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic Animals (MOZWE).Faculty of Veterinary Science,Mahidol University(Grant No.0517.131/5944)
文摘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.
基金supported by grants from the Ministry of Science and Technology,China(2011CB504702)National Natural Science Foundation of China(81290342)Development Grant of State Key Laboratory for Infectious Disease Prevention and Control(2008SKLID105)
文摘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.
基金supported by grants from the National Key Research and Development Program[2016YFD0500401]Development Grant of State Key Laboratory of Infectious Disease Prevention and Control[2015SKLID505,2014SKLID03]
文摘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.
基金supported by a grant from the Out-standing Person Innovation Foundation of Henan,China(0621002100)
文摘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.
基金supported by Important National Science & Technology Specific Projects (2012ZX10004403, 2012ZX10004219)National Natural Scientific Fund of China (81072675)
文摘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.
基金The Knowledge Innovation Program Key Project (KSCX1-YW-R-07)
文摘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.
文摘[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 bp) 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 western 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.
基金Supported by Youth Fund of Hubei Academy of Agricultural Sciences(2013NKYJJ12)
文摘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.
基金Financial support from Indian Council of Medical Research.New DelhiCouncil of Scientific,Industrial Research,New Delhi
文摘Objective:To report high co-positivity of anti-dengue virus(DV)and anti-Japanese encephalitis virus(JEV)IgM in an area endemic for both the viruses and to discuss the possibilities of coinfection.Methods:Serum samples from the patients who presented with fever,suspected central nervous system infection and thrombocytopenia,were tested for anti-DV IgM and antiJEV IgM antibodies.Conventional reverse transcriptase polymerase chain reaction was done for detection of DV RNA and JEV RNA.Results:Of 1 410 patient sera tested for anti-DV and antiJEV antibodies,129(9.14%)were co-positive for both.This co-positivity was observed only in those months when anli-JEV IgM positivily was high.Tilers of both anli-DV IgM and anti-JEV IgM were high in most of the co-positive eases.Among these 129 co-positive cases,76 were lesled by conventional reverse Iranscriplase polymerase chain reaction for both flaviviruses,of which eight cases were co-positive for DV and JEV.Conclusions:Co-infection with more than one fluvivirus species can occur in hyperendemic areas.
基金Indian Council of Madical Research,Government of India for sponsoring and funding the project
文摘Objective:To assess the virus infection in mosquitoes during different seasons and correlated with various climatic factors.Methods:The field collected vectors were screened for Japanese encephalitis(JE) virus after dessication using ELISA method.Most of the positive pools were recorded from Culex tritaeniorhynchus(Cx.tritaeniorhynchus) and Culex.gelidus(Cx.gelidus) during JE transmission season(winter) and some positive pools were also reported during non JE transmission periods(i.e.summer and rainy seasons).Results:The minimum infection rates(MIR) of 1.75 from Cx.tritaeniorhynchus and 0.17 from Cx.gelidus has been reported in the year 2002 at the beginning of the study and the values were found nil at the end of the study(2006) from the study areas of Kurnool district.Conclusions:From this study it is noted that MTR of Cx.gelidus and Cx.tritaeniorhynchus were modulated by various meteorological parameters.The mosquito vector abundance increases after the monsoon period(winter) and lowest in dry season(summer). Similarly,MIR fluctuated between seasons with higher MIR recorded after monsoon period and lower in the rest of season.Impact of these metrological parameters in JE virus infected mosquitoes is discussed in this paper.
文摘To investigate the potential utility of nuclease--resistant oligodeoxynucleotides (S-ODN) as anew class of antiviral agents. Methods: Two antisense phosphorothioate analogues (20--iner) complementary to thesequences of the first AUG codon and 5’ terminus of NSS of JEV SA--14 genome have been synthesized and their effects on CPE, viral antigen expression and virus plaque formation were tested in vitro. Results: The resultsshowed that 1. 0 pmol/L of S-ODN greatly deferred the onset of CPE in cultured BHKZI cells for at least 48 h.Addition of 5. 0 pmol/L or more S--ODN to culture medium fluid, 2 h prior to 100TCID,,virus inoculum, notablysuppressed viral antigen expression in the cells by making it lower than the limit of EIA detection in 48 h. The inhibition lasted more than 96 h. Viral plaque assay results demonstrated that S-ODN were most effect’ive within 18h with plaque inhibition rate over 90% by 5. 0 pmol/L S--ODNI. The inhibitory activity soon faded in 24 h. In addition, high concentrations (up to 80. 0 pmol/L) of S--ODN did not show any obvious cytotoxicity in 6 d by usingtrypan blue dye exclusion method. Conclusion: The specific synthetic S--ODN transitorily inhibited JEV replicationin BHK--ZI cells with characteristics of specificity and S--ODN dose--dependence.
基金This research was supported by a grant for project research from high Technology center of Kanazawa Medical University(H2000 2)
文摘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.
基金supported by the National Natural Science Foundation of China (30771067)
文摘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 nucleotide 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).
文摘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 per- sistently 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-PRSMTM310 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→Ser, 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 con- cluded 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 persis- tent infection of Japanese encephalitis virus.
基金supported by the Chinese National Science Foundation(39880032).
文摘A low-density oligonucleotide microarray was used for the detection of Japanese encephalitis virus (JEV) , combining with restriction display PCR labeling method. The hybridization targets were amplified from 6 plasmids containing several JEV gene fragments. Corresponding oligonucleotide probe spots were detected unambiguously. We claim that the oligonucleotide microarray technology is feasible and may have potential for clinical laboratory application.
