Japanese Encephalitis Virus Strains Research Articles

Protection of Mice against Japanese Encephalitis Virus Group II Strain Infections by Combinations of Monoclonal Antibodies to Different Antigenic Domains on Glycoprotein E

A combination of at least three hemagglutination-inhibition-positive (HAI) and virus-specific (Hs) monoclonal antibodies (MAbs) to glycoprotein E (gpE) of Japanese encephalitis virus (JEV) fully protected (100%) mice against JEV strain 733913 infections (group 1). However, these representative epitopes are reported to have been lost on JEV group II strains. In the present study, therefore, the protective effect of various combinations of anti-gpE MAbs representing antigenic epitopes other than Hs was studied on mice infections with JEV group II strains: JEV strains 641686 and 691004. MAbs used in the protective experiments were characterized as HAI-negative virus-specific (NHs) and HAI-positive flavivirus cross-reactive (Hx). Additionally, one of the Hs MAbs (MAb Hs-3) was included in the experiments. Mice were first administered single MAbs or their combinations intraperitoneally and 24 h later, infected with the virus intracerebrally. Protection rates of 70–75% were obtained with a combination of four MAbs: MAbs NHs-1, Hx-1, Hx-3 and Hs-3. However, protection rates of only 20–40% were obtained with three MAbs but none was observed with single or two MAbs. There was, however, a substantial increase in mice survival. The protective effect of several combinations of anti-gpE MAbs representing different antigenic epitopes might be due to the enhancement of binding within the same group and also between different MAb groups. The present results indicate that NHs and Hx epitopes should be incorporated with three Hs epitopes in a JEV vaccine that would have an added advantage, particularly in the flaviviral endemic areas with JEV strain variations.

Japanese encephalitis virus RNA detected in Culex pipiens mosquitoes in Italy

Mosquitoes collected in northern Italy were screened for flavivirus RNA. Positive amplicons were sequenced and found most similar to insect flavivirus (ISF), Usutu virus (USUV) and surprisingly also to Japanese encephalitis virus (JEV). The sequence (167 bp), obtained from one pool of Culex pipiens, was found identical to JEV strains from bats in China. Unfortunately additional sequence data or virus isolations were not obtained in this study. Confirmation of potential introduction of JEV to Italy and other European countries is urgently needed.

Pathogenic and vaccine strains of Japanese encephalitis virus elicit different levels of human macrophage effector functions

In India, Japanese encephalitis virus (JEV) remains one of the major causative agents of pediatric encephalitis. Macrophages support various neurotropic viruses and influence the immune response. However, the functional status of human macrophages during JEV infection remains unidentified. In this study, we examined the cytokine response and co-stimulatory marker levels in primary human monocyte derived macrophages (MDMs) infected with JE057434 (neurovirulent, primary clinical isolate) or SA14-14-2 (non-neurovirulent, live-attenuated vaccine) JEV strains. We also examined the differential susceptibility of these JEV strains to antiviral effects of interferon and nitric oxide. The results indicate that both JEV strains are capable of inducing various cytokines (type-I IFN, TNFα, IL6 and IL8) and co-stimulatory molecules (CD86 and CD80) in MDMs. However, they varied in replication potential and corresponding interferon sensitivity. SA14-14-2 was highly susceptible to interferon and nitric oxide when compared to JE057434. Thus, reduction in infectious virion production and increased sensitivity of SA14-14-2 towards interferon in MDMs could potentially play a role in limiting viral spread to additional target tissues.

Activated CD56+ lymphocytes (NK+NKT) mediate immunomodulatory and anti-viral effects during Japanese encephalitis virus infection of dendritic cells in-vitro

Japanese encephalitis virus (JEV) remains one of the major causative agents of pediatric encephalitis. Interaction of dendritic cells (DCs) with innate lymphocytes (NK and NKT) represents a crucial event during anti-viral innate immune response. In the current study, we have tried to understand the interaction between JEV, human monocyte derived DCs (MDDCs), and CD56+ cells (NK+NKT) in-vitro. We have used two JEV strains (i) JE057434 (neurovirulent, wild-type) and (ii) SA14-14-2 (non-neurovirulent, live-attenuated vaccine) to investigate the effect of viral virulence on the functional status of primary human MDDCs. Our preliminary results indicate that replicating JEV induces MDDCs maturation via PI3K and p38 pathways. We also show that the presence of IL2-activated CD56+ cells impart both immunomodulatory and anti-viral effects on DCs infected with JEV. Mechanistic studies illustrate that, IL2-activated CD56+ lymphocytes mediated immunomodulation occurs through direct cell-to-cell contact and TNFα, while the anti-viral effect is dependent on direct cell-to-cell contact.

Study on the molecular characteristics of Japanese encephalitis virus living in vector mosquitoes, in Zhejiang province, 2009 - 2010

To investigate the molecular characteristics of Japanese encephalitis virus (JEV) living in vector mosquitoes, from Zhejiang province. A total of 13 620 mosquitoes were collected from the monitoring stations located in Cixi city and Xianju county in Zhejiang province, in July and August, 2009 - 2010. Nucleic acid of JEV from the mosquitoes was monitored by using real-time RT-PCR. The virus strains were isolated with BHK-21 cell line, with E genes of the isolated viruses amplified, sequenced and their phylogeny and homology analyzed. The positive rates of JEV for those mosquitoes collected in the stations of Cixi and Xianju were 17.0% (27/159) and 3.4% (1/29), respectively. Twenty-two JEV strains were isolated, accounted for 15.4% among the 143 batches of mosquitoes collected in 2010. All E genes in the 6 sequenced virus isolates contained 1500 nucleotides encoding 500 amino acids, in which no inserts and deletions were identified. The identity rates of nucleotide and amino acid in E gene were 99.2% - 99.8% and 100.0% among the 6 JEV strains isolated from Zhejiang, 99.1% - 99.3% and 99.2% - 99.8% between the Zhejiang strains in 2009 - 2010 and the Zhejiang strains in 2007 - 2008, respectively, 87.6% - 88.0% and 97.8% between the 6 Zhejiang strains and the vaccine strain SA14-14-2 of JEV, respectively. The phylogeny tree of E gene indicated that the JEV isolates in Zhejiang during 2009 - 2010 was located in the branch of the genotype I. Mosquitoes collected from Cixi and Xianju areas carried JEV, with the rate of JEV in Cixi higher than in Xianju. All the Zhejiang isolates in 2009 - 2010 were proven to be the genotype I of JEV.

Distribution of Mosquitoes and Mosquito-Borne Viruses along the China-Myanmar Border in Yunnan Province

A total of 54,673 mosquitoes were collected at 11 sites located near the China-Myanmar border in the western part of Yunnan Province during July and August 2007. There were 29 species in 4 genera identified from the collections, including 12 species of Culex, 12 species of Anopheles, 3 species of Aedes, and 2 species of Armigeres. Culex tritaeniorhynchus Giles (67.9%, 37,119/54,673) and Anopheles sinensis Wiedemann (25.9%, 14,170/54,673) were the most abundant species in this investigation. Virus was isolated using BHK-21 and C6/36 cells from 22 of 510 mosquito pools. Isolates included Japanese encephalitis virus (JEV) and Getah virus (GETV), which were identified by serological and molecular methods. Twenty JEV strains were isolated from Cx. tritaeniorhynchus (15 isolates), An. sinensis (3 isolates), and Armigeres subalbatus Coquillett (2 isolates), and 2 GETV strains were isolated from Culex pseudovishnui Colless and Cx. tritaeniorhynchus. This study suggests that Ar. subalbatus is a potentially important local vector because of the high JEV infection ratio found in this species. Enzootic JEV transmission persists in this area and therefore, surveillance for human disease caused by JEV and GETV should be conducted in the region.

Molecular phylogenetic and evolutionary analysis of Japanese encephalitis virus in China

We elucidated the molecular epidemiology and evolution of Japanese encephalitis virus (JEV) strains isolated from 1949 to 2009 in China in this study. Three genotypes (I, III, V) were confirmed to be co-circulating in China in both high- and low-prevalence areas. Genotype III consisted of two clades (mainland clade and Taiwan clade). Compared to the mainland clade, genotype I and the Taiwan clade were newly introduced and evolved more rapidly. We also demonstrated that JEV strains in China, especially those in the mainland clade, were not only under purifying selection, but also probably under positive selection (aa 227 and 408 in the envelope protein).

Identification and characterization of the short variable region of the Japanese encephalitis virus 3′ NTR

Since the 1980s, the Japanese encephalitis virus (JEV) variants with slightly short variable regions (VR) of the 3' non-translated region (NTR) have been found; however, the implications of these short VR remain unclear. We recently identified two novel types of short VR (5 and 9 nt shorter than that of major group of genotype I JEV strains) of genotype I JEV isolates. To elucidate the impact of these short VR on the replication and virulence of JEV, we generated five recombinant JEV viruses: M41-d5 and M41-d9 have deletions in the VR that correspond to those observed in some recent JEV isolates, M41-d5d9 has both the 5- and 9-nt deletions in the VR, M41-d27 has a large deletion that encompasses both the 5- and 9-nt deletion regions, and M41-a13 has a 13-nt sequence insertion of the genotype III JEV strain Beijing-1 into the parent genotype I JEV strain Mie/41/2002 genome. The recombinant viruses and the parent virus, except for the M41-d27 mutant, showed similar growth properties in mammalian and mosquito cell lines. Mouse challenge experiments indicated that no significant differences among the recombinant viruses M41-d5d9, M41-d27, M41-a13, and the parent virus. Our results suggest that the short VR in JEV 3' NTR do not affect its growth in vitro or its pathogenicity in mice.

Broad-Spectrum Antiviral Activity of RNA Interference against Four Genotypes of Japanese Encephalitis Virus Based on Single MicroRNA Polycistrons

Japanese encephalitis virus (JEV), a neurotropic mosquito-borne flavivirus, causes acute viral encephalitis and neurologic disease with a high fatality rate in humans and a range of animals. Small interfering RNA (siRNA) is a powerful antiviral agent able to inhibit JEV replication. However, the high rate of genetic variability between JEV strains (of four confirmed genotypes, genotypes I, II, III and IV) hampers the broad-spectrum application of siRNAs, and mutations within the targeted sequences could facilitate JEV escape from RNA interference (RNAi)-mediated antiviral therapy. To improve the broad-spectrum application of siRNAs and prevent the generation of escape mutants, multiple siRNAs targeting conserved viral sequences need to be combined. In this study, using a siRNA expression vector based on the miR-155 backbone and promoted by RNA polymerase II, we initially identified nine siRNAs targeting highly conserved regions of seven JEV genes among strains of the four genotypes of JEV to effectively block the replication of the JEV vaccine strain SA14-14-2. Then, we constructed single microRNA-like polycistrons to simultaneously express these effective siRNAs under a single RNA polymerase II promoter. Finally, these single siRNAs or multiple siRNAs from the microRNA-like polycistrons showed effective anti-virus activity in genotype I and genotype III JEV wild type strains, which are the predominant genotypes of JEV in mainland China. The anti-JEV effect of these microRNA-like polycistrons was also predicted in other genotypes of JEV (genotypes II and IV), The inhibitory efficacy indicated that siRNAs×9 could theoretically inhibit the replication of JEV genotypes II and IV.

Isolation and molecular characterization of genotype 1 Japanese encephalitis virus, SX09S-01, from pigs in China

BackgroundPigs play a critical role in Japanese encephalitis virus (JEV) transmission between mosquitos and humans. In 2009, lots of piglets developed symptom of viral encephalitis in a pig farm in Yunchen, Shanxi province.MethodsVirus isolation was carried out in BHK-21 cells. Immunohistochemistry, RT-PCR and indirect immunofluorescent assay were used to identify the newly isolated virus. The complete genome of one isolate (SX09S-01 strain) was sequenced and analyzed. Two phylogenetic trees were constructed on the basis of the 24 full-length JEV genomes and 62 E genes mostly selected from China.ResultsJEV SX09S-01 strain was isolated from piglets. Sequence analysis indicates that the completed genome sequences of this strain consists of 10965 nucleotides and there are 13 nucleotides deletion in the 3' nontranslated variable region. Compared with other JEV strains, homology ranges from 99.1% (XJ69) to 74.1% (XZ0934) and 99.6% (XJ69) to 91.1% (XZ0934) on the level of nucleotide and amino acid sequences, respectively. Phylogenetic trees show that SX09S-01 strain belongs to genotype I and it is most closely related to the XJ69 strain.ConclusionsGenotype I of JEV still circulates in Yuncheng and it is thus important for active surveillance on genotype I of JEV from the swine population.

Serological and molecular diagnosis of Japanese encephalitis reveals an increasing public health problem in the state of West Bengal, India

Japanese encephalitis (JE), a neurotrophic disease, was first recorded in the state of West Bengal, India in 1973. Since then JE is being reported every year from different districts. With a view to identify the JE cases accurately, a study was undertaken to detect the Japanese encephalitis virus (JEV) as the etiologic agent from the acute encephalitis syndrome (AES) cases and to identify its distribution in different districts. We report the results of 513 blood or cerebrospinal fluid samples referred/collected from the hospitalized AES cases. The samples were initially subjected to Mac-ELISA test followed by reverse transcriptase (RT)-PCR for the detection of IgM antibodies and the JEV genome, specific to E gene, respectively. Out of 513 samples referred/collected, 139 (27.1%) samples were reactive to JE IgM antibody. The remaining 374 samples were screened to select those which had a history of illness with a duration of ≤3 days. Only 147 samples were selected and tested, out of which 36 (24.5%) isolates were achieved and those were RT-PCR positive against the control JEV strain. Detection of IgM antibody to JE and the RT-PCR result confirms the active circulation of JEV in different districts of West Bengal and needs to be monitored carefully.

Autophagy is involved in the early step of Japanese encephalitis virus infection

Japanese encephalitis virus (JEV), an enveloped Flavivirus with a positive-sense RNA genome, causes acute encephalitis with high mortality in humans. We used a virulent (RP-9) and an attenuated (RP-2ms) JEV strain to assess the role of autophagy in JEV infection. By monitoring the levels of lipidated LC3, we found that autophagy was induced in human NT-2 cells infected with RP-2ms, especially at the late stage, and to a lesser extent with RP-9. The induction of autophagy by rapamycin increased viral production, whereas the inhibition of autophagy by 3-methyladenine reduced viral yields for both RP-9 and RP-2ms. The viral replication of RP-9 and RP-2ms was also reduced in cells with downregulated ATG5 or Beclin 1 expression, suggesting a proviral role of autophagy in JEV replication. To determine the step of JEV life cycle affected by autophagy, we used an mCherry−LC3 fusion protein as the autophagosome marker. Little of no colocalization of LC3 puncta with dsRNA was noted, whereas the input JEV particles were targeted to autophagosomes stained positive for early endosome marker. Overall, we show for the first time that the cellular autophagy process is involved in JEV infection and the inoculated viral particles traffic to autophagosomes for subsequent steps of viral infection.

Simultaneous detection and differentiation of dengue virus serotypes 1-4, Japanese encephalitis virus, and West Nile virus by a combined reverse-transcription loop-mediated isothermal amplification assay

BackgroundRapid identification and differentiation of mosquito-transmitted flaviviruses in acute-phase sera of patients and field-caught vector mosquitoes are important for the prediction and prevention of large-scale epidemics.ResultsWe developed a flexible reverse-transcription loop-mediated isothermal amplification (RT-LAMP) unit for the detection and differentiation of dengue virus serotypes 1-4 (DENV1-4), Japanese encephalitis virus (JEV), and West Nile virus (WNV). The unit efficiently amplified the viral genomes specifically at wide ranges of viral template concentrations, and exhibited similar amplification curves as monitored by a real-time PCR engine. The detection limits of the RT-LAMP unit were 100-fold higher than that of RT-PCR in 5 of the six flaviviruses. The results on specificity indicated that the six viruses in the assay had no cross-reactions with each other. By examining 66 viral strains of DENV1-4 and JEV, the unit identified the viruses with 100% accuracy and did not cross-react with influenza viruses and hantaviruses. By screening a panel of specimens containing sera of 168 patients and 279 pools of field-caught blood sucked mosquitoes, results showed that this unit is high feasible in clinical settings and epidemiologic field, and it obtained results 100% correlated with real-time RT-PCR.ConclusionsThe RT-LAMP unit developed in this study is able to quickly detect and accurately differentiate the six kinds of flaviviruses, which makes it extremely feasible for screening these viruses in acute-phase sera of the patients and in vector mosquitoes without the need of high-precision instruments.

An amino acid substitution (V3I) in the Japanese encephalitis virus NS4A protein increases its virulence in mice, but not its growth rate in vitro

Our previous studies have shown that the Japanese encephalitis virus (JEV) strain Mie/40/2004 is the most virulent of the strains isolated by us in Japan from 2002 to 2004. Comparison of the amino acid sequence of Mie/40/2004 with those of low-virulence strains revealed that an isoleucine residue at position 3 of the Mie/40/2004 NS4A protein may increase viral pathogenicity. A recombinant virus with a single valine-to-isoleucine substitution (V3I) at position 3 in the low-virulence Mie/41/2002 background (rJEV-Mie41-NS4A(V3I)) exhibited increased virulence in mice compared with the Mie/41/2002 parent strain. The V3I mutation did not affect virus growth in several cell lines. These results demonstrate that the isoleucine at position 3 in the NS4A protein of Mie/40/2004 is responsible for its high virulence in vivo. This is the first report to show that an amino acid substitution in a flavivirus NS4A protein alters viral pathogenicity in mice.

Correlation of protection against Japanese encephalitis virus and JE vaccine (IXIARO ®) induced neutralizing antibody titers

Immune sera from volunteers vaccinated in a blinded Phase 3 clinical trial with JE-VAX ® and a new Japanese encephalitis virus (JEV) vaccine (IC51 or IXIARO), were tested for the ability to protect mice against lethal JEV challenge. Sera from IXIARO vaccinated subjects were pooled into four batches based on neutralizing antibody measured by plaque reduction neutralization test (PRNT 50 titer): high (∼200), medium (∼40–50), low (∼20) and negative (<10). Pooled sera from JE-VAX ® vaccinated subjects (PRNT 50 titer ∼ 55) and pooled JEV antibody negative pre-vaccination sera were used as controls. Groups of ten 6- to 7-week-old female ICR mice were injected intraperitoneally with 0.5 ml of each serum pool diluted 1:2 or 1:10, challenged approximately 18 h later with a lethal dose of either JEV strain SA14 (genotype III) or strain KE-093 (genotype I) and observed for 21 days. All mice in the non-immune serum groups developed clinical signs consistent with JEV infection or died, whereas high titer sera from both IXIARO and JE-VAX ® sera protected 90–100% of the animals. Statistical tests showed similar protection against both JEV strains SA14 and KE-093 and protection correlated with the anti-JEV antibody titer of IXIARO sera as measured by PRNT 50. Ex vivo neutralizing antibody titers showed that almost all mice with a titer of 10 or greater were fully protected. In a separate study, analysis of geometric mean titers (GMTs) of the groups of mice vaccinated with different doses of IXIARO and challenged with JEV SA14 provided additional evidence that titers ≥ 10 were protective.

Vimentin binding is critical for infection by the virulent strain of Japanese encephalitis virus

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes acute encephalitis with high mortality in humans. We used a pair of virulent (RP-9) and attenuated (RP-2ms) variants of JEV to pull down the cell surface molecules bound with JEV particle; their identities were revealed by LC-MS/MS analysis. One major protein bound with RP-9 and weakly with RP-2ms was identified as the intermediate filament protein vimentin. Infection of RP-9 but not that of RP-2ms was blocked by anti-vimentin antibodies and by recombinant-expressed vimentin proteins. Knockdown of vimentin expression reduced the levels of viral binding and viral production of RP-9, but not that of RP-2ms. The different vimentin dependency for JEV infection could be attributed to the major structural envelope protein, as the recombinant RP-9 with an E-E138K mutation became resistant to anti-vimentin blockage. Furthermore, RP-2ms mainly depended on cell surface glycosaminoglycans for viral binding and it became vimentin-dependent only when binding to glycosaminoglycans was blocked. Thus, we suggest that vimentin contributes to virulent JEV infection and might be a new target to intervene in this deadly infection.

Characterization of nonstructural protein 3 of a neurovirulent Japanese encephalitis virus strain isolated from a pig

BackgroundJapanese encephalitis virus (JEV), as a re-emerging virus that causes 10,000-15,000 human deaths from encephalitis in the world each year, has had a significant impact on public health. Pigs are the natural reservoirs of JEV and play an important role in the amplification, dispersal and epidemiology of JEV. The nonstructural protein 3 (NS3) of JEV possesses enzymatic activities of serine protease, helicase and nucleoside 5'-triphosphatase, and plays important roles in viral replication and pathogenesis.ResultsWe characterized the NS3 protein of a neurovirulent strain of JEV (SH-JEV01) isolated from a field-infected pig. The NS3 gene of the JEV SH-JEV01 strain is 1857 bp in length and encodes protein of approximately 72 kDa with 99% amino acid sequence identity to that of the representative immunotype strain JaGAr 01. The NS3 protein was detectable 12 h post-infection in a mouse neuroblastoma cell line, Neuro-2a, and was distributed in the cytoplasm of cells infected with the SH-JEV01 strain of JEV. In the brain of mice infected with the SH-JEV01 strain of JEV, NS3 was detected in the cytoplasm of neuronal cells, including pyramidal neurons of the cerebrum, granule cells, small cells and Purkinje cells of the cerebellum.ConclusionsThe NS3 protein of a neurovirulent strain of JEV isolated from a pig was characterized. It is an approximately 72 kDa protein and distributed in the cytoplasm of infected cells. The Purkinje cell of the cerebellum is one of the target cells of JEV infection. Our data should provide some basic information for the study of the role of NS3 in the pathogenesis of JEV and the immune response.

Continuity and Change of Japanese Encephalitis Virus in Toyama Prefecture, Japan

To determine the mechanisms of maintenance and evolution of Japanese encephalitis virus (JEV) in a temperate zone, we attempted to isolate JEV from mosquitoes and pigs in Toyama Prefecture, Japan. A total of 87 JEVs were isolated from female Culex tritaeniorhynchus mosquitoes and pigs during 2005-2009. The prevalence of JEV in Toyama Prefecture was seasonally late in comparison with that of the virus during 1966-1972. Furthermore, JEVs were isolated after the peak in the number of female Cx. tritaeniorhynchus. Among JEV strains isolated in this study, two distinct groups were observed within genotype I of the phylogeny generated from nucleotide sequence information derived from the envelope and capsid/premembrane genes: strains belonging to the major type were isolated during 2005-2009, and strains from the minor type were isolated only in 2007. The major type has exhibited gradual change in its envelope and capsid/premembrane genes, and all isolates obtained in 2008 and 2009 had a novel deletion of seven nucleotides in the variable region of the 3'-untranslated region.

Japanese Encephalitis, Tibet, China

Japanese Encephalitis, Tibet, China

Delineation of an epitope on domain I of Japanese Encephalitis Virus envelope glycoprotein using monoclonal antibodies

The Envelope glycoprotein (E-protein) of Japanese encephalitis virus (JEV) is the major structural component on the virion surface and is a primary target for the host immune system. Two monoclonal antibodies (MAbs) NHA-I (IgG2b) and NHA-II (IgM) against JEV (Indian strain 733913) were earlier developed in the authors’ laboratory and found to be cross-reactive to nuclear histones. However, the epitope specificity of these MAbs has remained unknown. The present study was carried out to delineate the epitopes recognised by these MAbs on the E-protein of JEV strain 733913. The variable regions of the NHA-I and NHA-II were sequenced and the tertiary structures predicted. Molecular docking of the MAbs with the structural model of the JEV E-protein demonstrated that NHA-I binds to a predicted antigenic determinant (residue position 18–33) in domain-I. To understand the epitope specificity and check for possible cross-reactivity of these MAbs, comparative analysis of interactions with the known crystallographic structure of the West Nile virus (WNV) E-protein was also carried out. The studies predicted a differential binding of NHA-I but not of NHA-II between JEV and WNV. Mutagenesis studies could help analyse the specificity of NHA-I. The NHA-II appears to be cross-reactive as it docked in the groove region between domains I and III of both the JEV and WNV E-proteins. In laboratory assays, namely, ELISA and immunofluorescence assay both the MAbs reacted equally with JEV while the NHA-I did not show any reactivity with WNV. In silico results were thus validated by laboratory experiments. The present study would help in better understanding of virus–host interactions at the molecular level, and also be useful for the future design of vaccines as well as peptide based diagnostics.