Japanese Encephalitis and Associated Environmental Risk Factors in Eastern Uttar Pradesh: A time series analysis from 2001 to 2016.

India and other Southeast Asian countries are severely affected by Japanese encephalitis (JE), one of the deadliest vector-borne disease threat to human health. Several epidemiological observations suggest climate variables play a role in providing a favorable environment for mosquito development and virus transmission. In this study, generalized additive models were used to determine the association of JE admissions and mortality with climate variables in Gorakhpur district, India, from 2001-2016. The model predicted that every 1 unit increase in mean (Tmean;°C), and minimum (Tmin;°C) temperature, rainfall (RF; mm) and relative humidity (RH; %) would on average increase the JE admissions by 22.23 %, 17.83 %, 0.66 %, and 5.22 % respectively and JE mortality by 13.27 %, 11.77 %, 0.94 %, and 3.27 % respectively Conversely, every unit decrease in solar radiation (Srad; MJ/m2/day) and wind speed (WS; Kmph) caused an increase in JE admission by 17% and 11.42% and in JE mortality by 9.37% and 4.88% respectively suggesting a protective effect at higher levels. The seasonal analysis shows that temperature was significantly associated with JE in pre-monsoon and post-monsoon while RF, RH, Srad, and WS are associated with the monsoon. Effect modification due to age and gender showed an equal risk for both genders and increased risk for adults above 15 years of age, however, males and age groups under 15 years outnumbered females and adults. Sensitivity analysis results to explore lag effects in climate variables showed that climate variables show the strongest association at lag 1 to 1.5 months with significant lag effect up tp lag 0-60 days. The exposure-response curve for climate variables showed a more or less linear relationship, with an increase in JE admissions and mortality after a certain threshold and decrease were reported at extreme levels of exposure. The study concludes that climate variables could influence the JE vector development and multiplication and parasite maturation and transmission in the Gorakhpur region whose indirect impact was noted for JE admission and mortality. In response to the changing climate, public health interventions, public awareness, and early warning systems would play an unprecedented role to compensate for future risk.

The spatiotemporal distribution of Japanese Encephalitis cases in Yunnan Province, China, from 2007 to 2017.

BACKGROUND: Japanese encephalitis (JE) is a vector-borne disease with a high prevalence in Yunnan Province, China. However, there has been a lack of a JE epidemic systematic analysis, which is urgently needed to guide control and prevention efforts. METHODS: This study explored and described the spatiotemporal distribution of JE cases observed among two different age groups in Yunnan Province from 2007 to 2017. The epidemiological features and spatial features were analyzed according to basic statistics, ArcGIS software (version 9.3; ESRI, Redlands, CA) and SPSS software (version 20; IBM Corp., Armonk, New York). RESULTS: Overall, the whole province had a high incidence of JE. The annual incidence rates in 2007 and 2017 were 1.668/100,000 and 0.158/100,000, respectively. The annual mortality was under 0.095/100,000 for these years. Although the whole province was in danger of JE, the Diqing autonomous prefecture and the Lijiang autonomous prefecture had no JE cases recorded for over 10 years. The JE cases were reported by hospitals located in 60 counties of 14 municipalities. The top ten areas with the most JE cases were Kunming City, Zhaotong City, Jinghong City, Wenshan City, Mangshi City, Pu'er City, Baoshan City, Dali City, Chuxiong City, and Gejiu City. The incidence declined smoothly, with a peak occurring from June to September, which accounted for 96.1% of the total cases. Children whose age was equal or less than 10 years old (LEQ10) still maintained a high frequency of JEV infection, and a large number of cases were reported in August, despite the Expanded Program on Immunization (EPI), which was established in April 2008. There was no difference in the quantity of cases between the two groups (t = -0.411, P>0.05); additionally, the number of JE cases among patients LEQ10 were significantly greater than those among patients older than 10 years (GTR10). Further analysis using local indicators of spatial association (LISA) revealed that the distribution of JE exhibited a high-high cluster characteristic (Z = 2.06, P<0.05), which showed that Jinghong City, Guangnan County, Yanshan County, Funing County, and Mengzi City were hot spots for the JE epidemic. CONCLUSIONS: Although the EPI was established in 2008 and the incidence of JE declined smoothly in Yunnan Province, there was no difference in the number of cases between the two age groups, which reveals that the EPI has been conducted with a low level success. In the context of limited vaccine supply capacity, we should strengthen the implementation of the children's immunization program before strengthening other immunization programs.

Japanese encephalitis virus neuropenetrance is driven by mast cell chymase.

Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis. However, the mechanisms of JEV penetration of the blood-brain-barrier (BBB) remain poorly understood. Mast cells (MCs) are granulated innate immune sentinels located perivascularly, including at the BBB. Here we show that JEV activates MCs, leading to the release of granule-associated proteases in vivo. MC-deficient mice display reduced BBB permeability during JEV infection compared to congenic wild-type (WT) mice, indicating that enhanced vascular leakage in the brain during JEV infection is MC-dependent. Moreover, MCs promoted increased JEV infection in the central nervous system (CNS), enhanced neurological deficits, and reduced survival in vivo. Mechanistically, chymase, a MC-specific protease, enhances JEV-induced breakdown of the BBB and cleavage of tight-junction proteins. Chymase inhibition reversed BBB leakage, reduced brain infection and neurological deficits during JEV infection, and prolonged survival, suggesting chymase is a novel therapeutic target to prevent JEV encephalitis.

Possible pathogenicity of Japanese encephalitis virus in newly hatched domestic ducklings.

Japanese encephalitis virus (JEV) is a zoonotic flavivirus that is transmitted by mosquitoes and vertebrate-amplifying hosts, including birds. Domestic ducks are susceptible to JEV infection and develop various levels of viremia. We tested the pathogenicities of seven JEV strains in newly hatched domestic ducklings. All inoculated ducklings showed stunted growth. Two JEV strains caused notable mortalities of 12.7% and 31.7%, respectively, highlighting that some emerged JEV strains may thus be pathogenic in newly hatched domestic ducklings.

Effective inhibition of different Japanese encephalitis virus genotypes by RNA interference targeting two conserved viral gene sequences in vitro and in vivo.

Japanese encephalitis is a zoonotic, mosquito-borne, infectious disease caused by Japanese encephalitis virus (JEV), which is prevalent in China. At present, there are no specific drugs or therapies for JEV infection, which can only be treated symptomatically. Lentivirus-mediated RNA interference (RNAi) is a highly efficient method to silence target genes. In this study, two lentiviral shRNA, LV-C and LV-NS5, targeting the conserved viral gene sequences were used to inhibit different JEV genotypes strains in BHK21 cells and mice. The results showed that LV-C significantly inhibited JEV genotype I and genotype III strains in cells and mice. Quantitative RT-PCR analysis showed that JEV mRNA were reduced by 83.2-90.9% in cells by LV-C and that flow cytometry analysis confirmed the inhibitory activity of LV-C. The viral titers were reduced by about 1000-fold in cells and the brains of suckling mice by LV-C, and the pretreatment of LV-C protected 60-80% of mice against JEV-induced lethality. The inhibitory activities of LV-NS5 in cells and mice were weaker than those of LV-C. These results indicate that RNAi targeting of the two conserved viral gene sequences had significantly suppressed the replication of different JEV genotypes strains in vitro and in vivo, highlighting the feasibility of RNAi targeting of conserved viral gene sequences for controlling JEV infection.

Entomological investigation of Japanese encephalitis outbreak in Malkangiri district of Odisha state, India.

BACKGROUND: A severe outbreak of Japanese encephalitis (JE) and acute encephalitis syndrome (AES) with high case fatality was reported from Malkangiri district of Odisha state, India during September to November 2016 affecting 336 children with 103 deaths. OBJECTIVES: The purpose of this study was to investigate the outbreak in the light of entomological determinants. METHODS: Entomological investigation was carried out in 48 villages from four mostly affected Community Health Centres (CHCs) of Malkangiri district. Dusk collections of resting adults was done in villages from indoor and outdoor sites to record the density of mosquito species, including the known JE vectors, feeding behaviour, parity, dusk index and infection status with JE virus (JEV). FINDINGS: The per man hour density and dusk index of JE vector species varied from 2.5 to 24.0 and 0.81 to 7.62, respectively in study villages. A total of 1136 mosquitoes belonging to six vector species were subjected to PCR and one pool of Culex vishnui was found to be positive for JEV. CONCLUSION: The JE transmission in Malkangiri district was confirmed. Thorough screening of human blood samples of JE/AES suspected cases and JE vector mosquitoes for the presence of JEV during rainy season every year is recommended.

A Simple Mechanism Based on Amino Acid Substitutions is not a Critical Determinant of High Mortality of Japanese Encephalitis Virus Infection in Mice.

For the development of effective treatment strategies for Japanese encephalitis (JE), it is important to identify the viral factors causing severe disease during JE virus (JEV) infection. In this study, we assessed whether amino acid substitutions are critical factors for higher mortality of JaTH160 compared with JaOArS982 in mice using the technique of infectious cDNA clones. We raised the possibility that two amino acids of C124 and NS3482 of JaTH160 may contribute to increased mortality in mice. However, simultaneous substitutions of these amino acids did not significantly increase the virulence of JaOArS982, suggesting that high mortality due to JaTH160 viral infection cannot be simply attributed to the specific amino acids. Multiple and complex, but not simple, mechanisms may induce the high mortality of JaTH160 infection in mice.

Spatio-temporal epidemiology of Japanese encephalitis in Nepal, 2007-2015.

Japanese encephalitis (JE) is a major public health problem in Nepal. For the effective management and surveillance of JE, a clear understanding of its epidemiology is essential. Therefore, we conducted descriptive and spatial analyses to understand the spatio-temporal distribution of JE in human in Nepal. From 2007 to 2015, 1,823 JE cases were reported with a cumulative mean incidence of 0.735/100,000 population and a case fatality rate of 6.6%. The death rate in the up-to-24 years of age group was 74%. The JE cases were most commonly reported in the age group of 1-14 years. There is a strong seasonal pattern of JE occurrence in Nepal which peaked in August and declined by October each year, which corresponds to the monsoon season. The JE cases were reported in 63 of 75 districts (84%), expanding in the mountain and hill regions. There was a strong clustering of JE incidence in the south-western and south-eastern Terai region, which is endemic for JE. Therefore, the JE surveillance system should be improved to better understand the drivers of disease expansion in Nepal for instituting a control program.

Near-atomic structure of Japanese encephalitis virus reveals critical determinants of virulence and stability.

Although several different flaviviruses may cause encephalitis, Japanese encephalitis virus is the most significant, being responsible for thousands of deaths each year in Asia. The structural and molecular basis of this encephalitis is not fully understood. Here, we report the cryo-electron microscopy structure of mature Japanese encephalitis virus at near-atomic resolution, which reveals an unusual "hole" on the surface, surrounded by five encephalitic-specific motifs implicated in receptor binding. Glu138 of E, which is highly conserved in encephalitic flaviviruses, maps onto one of these motifs and is essential for binding to neuroblastoma cells, with the E138K mutation abrogating the neurovirulence and neuroinvasiveness of Japanese encephalitis virus in mice. We also identify structural elements modulating viral stability, notably Gln264 of E, which, when replaced by His264 strengthens a hydrogen-bonding network, leading to a more stable virus. These studies unveil determinants of neurovirulence and stability in Japanese encephalitis virus, opening up new avenues for therapeutic interventions against neurotropic flaviviruses.Japanese encephalitis virus (JEV) is a Flavivirus responsible for thousands of deaths every year for which there are no specific anti-virals. Here, Wang et al. report the cryo-EM structure of mature JEV at near-atomic resolution and identify structural elements that modulate stability and virulence.

CD8 T cells protect adult naive mice from JEV-induced morbidity via lytic function.

Following Japanese encephalitis virus (JEV) infection neutralizing antibodies are shown to provide protection in a significant proportion of cases, but not all, suggesting additional components of immune system might also contribute to elicit protective immune response. Here we have characterized the role of T cells in offering protection in adult mice infected with JEV. Mice lacking α/ß-T cells (TCRß-null) are highly susceptible and die over 10-18 day period as compared to the wild-type (WT) mice which are resistant. This is associated with high viral load, higher mRNA levels of proinflammatory cytokines and breach in the blood-brain-barrier (BBB). Infected WT mice do not show a breach in BBB; however, in contrast to TCRß-null, they show the presence of T cells in the brain. Using adoptive transfer of cells with specific genetic deficiencies we see that neither the presence of CD4 T cells nor cytokines such as IL-4, IL-10 or interferon-gamma have any significant role in offering protection from primary infection. In contrast, we show that CD8 T cell deficiency is more critical as absence of CD8 T cells alone increases mortality in mice infected with JEV. Further, transfer of T cells from beige mice with defects in granular lytic function into TCRß-null mice shows poor protection implicating granule-mediated target cell lysis as an essential component for survival. In addition, for the first time we report that γ/δ-T cells also make significant contribution to confer protection from JEV infection. Our data show that effector CD8 T cells play a protective role during primary infection possibly by preventing the breach in BBB and neuronal damage.

Mesenchymal stem cells alleviate Japanese encephalitis virus-induced neuroinflammation and mortality.

BACKGROUND: Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia. Japanese encephalitis (JE) caused by JEV is characterized by extensive inflammatory cytokine secretion, microglia activation, blood-brain barrier (BBB) breakdown, and neuronal death, all of which contribute to the vicious cycle of inflammatory damage. There are currently no effective treatments for JE. Mesenchymal stem cells (MSCs) have been demonstrated to have a therapeutic effect on many central nervous system (CNS) diseases by regulating inflammation and other mechanisms. METHODS: In vivo, 8- to 10-week-old mice were infected intraperitoneally with JEV and syngeneic bone marrow MSCs were administered through the caudal vein at 1 and 3 days post-infection. The mortality, body weight, and behavior were monitored daily. Brains from each group were harvested at the indicated times for hematoxylin and eosin staining, immunohistochemical observation, flow cytometric analysis, TUNEL staining, Western blot, quantitative real-time polymerase chain reaction, and BBB permeability assays. In vitro, co-culture and mixed culture experiments of MSCs with either microglia or neurons were performed, and then the activation state of microglia and survival rate of neurons were tested 48 h post-infection. RESULTS: MSC treatment reduced JEV-induced mortality and improved the recovery from JE in our mouse model. The inflammatory response, microglia activation, neuronal damage, BBB destruction, and viral load (VL) were significantly decreased in the MSC-treated group. In co-culture experiments, MSCs reprogrammed M1-to-M2 switching in microglia and improved neuron survival. Additionally, the VL was decreased in Neuro2a cells in the presence of MSCs accompanied by increased expression of interferon-α/ß. CONCLUSION: MSC treatment alleviated JEV-induced inflammation and mortality in mice.

Antisense Oligonucleotides Targeting Raf-1 Block Japanese Encephalitis Virus In Vitro and In Vivo.

Japanese encephalitis virus (JEV) infections represent a major health concern in Southeast Asia since no effective treatments are available. Recently, several reports have demonstrated that inhibition of certain host cell proteins prevents viral infection. Raf-1 kinase is a central component of many signaling pathways involved in normal cell growth and oncogenic transformation, and Ras/Raf/ERK signaling activation has been observed during viral infections (including JEV infection). In this study, Raf-1 was confirmed to be upregulated by JEV infection, which suggested that Raf-1 might be important for JEV infection and might be a target for novel anti-JEV drugs. To determine the role of Raf-1 during the JEV infection process, antisense oligonucleotides (ASODNs) were used to downregulate Raf-1 expression in JEV-infected baby hamster kidney (BHK-21) cells and African green monkey kidney (Vero) cells. From five ASODNs candidates tested, Raf-1-1 (Raf-1 antisense) significantly downregulated Raf-1 protein expression levels, significantly inhibited cytopathic effect (CPE) in cultured cells, and reduced JEV RNA levels in cell medium without affecting cell viability. Furthermore, it also demonstrated that ASODN Raf-1-1 possessed therapeutic effects by using a lethal JEV infection mouse model. In conclusion, data presented in this report demonstrated that ASODN Raf-1-1 could suppress Raf-1 protein and that Raf-1 inhibition suppressed JEV replication in vitro and in vivo. These data provided evidence for targeting Raf-1 in the development of novel anti-JEV therapies. In addition, Raf-1-1 represents potential drugs that can be adapted for treating JEV infections.

A simple method for developing an infectious cDNA clone of Japanese encephalitis virus.

Flavivirus cDNA clones frequently demonstrate genetic instability in transformed bacteria, which hampers the construction and manipulation of cDNAs for infectious flaviviruses. In this study, we developed a stable, full-length cDNA clone, pJEHEN, of a GI JEV strain HEN0701 using a medium-copy-number pBR322 vector and propagating cDNA clones at room temperature. The virus vJEHEN recovered from the infectious clone was indistinguishable from the parent virus HEN0701 with respect to plaque morphology, growth kinetics, and virulence characteristics. A T-to-A silent mutation of nucleotide 24 of the NS2a gene was introduced into the infectious cDNA clone to eliminate frameshifting. The rescued mutant virus vJETA did not express NS1' in infected cells and showed reduced growth and neurovirulence in mice. This convenient method for the construction and manipulation of infectious JEV cDNA clones may be of use in further studies to improve our understanding of the molecular mechanisms responsible for JEV replication and pathogenesis.

Can we differentiate between herpes simplex encephalitis and Japanese encephalitis?

BACKGROUND: Herpes simplex encephalitis (HSE) occurs without regional and seasonal predilections. HSE is important to differentiate from arboviral encephalitis in endemic areas because of therapeutic potential of HSE. This study evaluates clinical features, MRI and laboratory findings which may help in differentiating HSE from Japanese encephalitis (JE). METHODS: Confirmed patients with JE and HSE in last 10years were included. The presenting clinical symptoms including demographic information, seizure, behavioral abnormality, focal weakness and movement disorders were noted. Cranial MRI was done and location and nature of signal alteration were noted. Electroencephalography (EEG), cerebrospinal fluid (CSF), blood counts and serum chemistry were done. Outcome was measured by modified Rankin Scale (mRS). Death, functional outcome and neurological sequelae were noted at 3, 6 and 12months follow up, and compared between HSE and JE. Outcome was categorized as poor (mRS;>2) and good (mRS≤2). RESULTS: 97 patients with JE and 40 HSE were included. JE patients were younger than HSE and occurred in post monsoon period whereas HSE occurred throughout the year. Seizure (86% vs 40%) and behavioral abnormality (48% vs 10%) were commoner in HSE; whereas movement disorders (76% vs 0%) and focal reflex loss (42% vs 10%) were commoner in JE. CSF findings and laboratory parameters were similar in both the groups. Thalamic involvement in JE and temporal involvement in HSE were specific markers of respective encephalitis. Delta slowing on EEG was more frequent in JE than HSE. 20% JE and 30% HSE died in the hospital, and at 1year follow up JE patients showed better outcome compared to HSE (48% vs 24%). Memory loss (72% vs 22%) was the predominant sequelae in HSE. CONCLUSION: Seizure and behavioral abnormality are common features in HSE whereas focal reflex loss is commoner in JE. In a patient with acute encephalitis, thalamic lesion suggests JE and temporal lobe involvement HSE. Long term outcome in JE is better compared to HSE.

Enhanced immune responses against Japanese encephalitis virus using recombinant adenoviruses coexpressing Japanese encephalitis virus envelope and porcine interleukin-6 proteins in mice.

Japanese encephalitis is a reproductive disorder caused by Japanese encephalitis virus (JEV) in swine. Previous studies have demonstrated that recombinant adenovirus serotype 5 (Ad5) may be a potential vaccine candidate because it can express JEV envelope epitopes and induce immune responses against JEV. Still, it will be necessary to develop an adjuvant that can enhance both humoral and cellular immune responses to the recombinant antigen delivered by non-replicating Ad5. In this study, we investigated the systemic immune responses of BALB/c mice immunized with recombinant adenovirus expressing JEV envelope epitopes in combination with porcine interleukin-6 (rAdE-IL-6).The rAdE-IL-6 immunized group had the highest titers of anti-JEV antibody as detected by an enzyme-linked immunosorbent assay (ELISA), as well as the highest levels of neutralizing antibody (1:75) as detected by a serum neutralization test. Similarly, higher concentrations of interferon-gamma (834.7pg/ml) and interleukin-6 (IL-6) (229.7pg/ml) were detected in the rAdE-IL-6 group using an ELISA assay. These data indicate that immunized BALB/c induce a strong cellular response against rAdE-IL-6. Furthermore, after challenge with the virulent JEV SCYA201201 strain, the rAdE-IL-6 group generated an immune protective response 70% greater than that of the control group, indicating that rAdE-IL-6 induced a protective immune response against JEV challenge in mice. The results from this study demonstrated that IL-6 is a strong adjuvant that can enhance both humoral and cellular immune responses in mice. Furthermore, a recombinant adenovirus coexpressing JEV envelope epitopes and porcine IL-6 protein may be an effective vaccine in animals.

A large outbreak of Japanese encephalitis predominantly among adults in northern region of West Bengal, India.

Unusual rise of acute encephalitis syndrome cases (AES) were reported in July 2014 in the northern region of West Bengal, India. Investigations were carried out to characterize the outbreak and to identify the associated virus etiology. This observational study is based on 398 line listed AES cases, mostly (70.8%, 282/398) adults, with case fatality ratio of 28.9% (115/398). Japanese encephalitis virus infection was detected in 134 (49.4%) among 271 AES cases tested and most of them (79.1%, 106/134) were adults. The study reports a large outbreak of genotype III Japanese encephalitis among adults in northern region of West Bengal, India. J. Med. Virol. 88:2004-2011, 2016. © 2016 Wiley Periodicals, Inc.

MicroRNA-19b-3p Modulates Japanese Encephalitis Virus-Mediated Inflammation via Targeting RNF11.

UNLABELLED: Japanese encephalitis virus (JEV) can invade the central nervous system and consequently induce neuroinflammation, which is characterized by profound neuronal cell damage accompanied by astrogliosis and microgliosis. Albeit microRNAs (miRNAs) have emerged as major regulatory noncoding RNAs with profound effects on inflammatory response, it is unknown how astrocytic miRNAs regulate JEV-induced inflammation. Here, we found the involvement of miR-19b-3p in regulating the JEV-induced inflammatory responsein vitroandin vivo The data demonstrated that miR-19b-3p is upregulated in cultured cells and mouse brain tissues during JEV infection. Overexpression of miR-19b-3p led to increased production of inflammatory cytokines, including tumor necrosis factor alpha, interleukin-6, interleukin-1ß, and chemokine (C-C motif) ligand 5, after JEV infection, whereas knockdown of miR-19b-3p had completely opposite effects. Mechanistically, miR-19b-3p modulated the JEV-induced inflammatory response via targeting ring finger protein 11, a negative regulator of nuclear factor kappa B signaling. We also found that inhibition of ring finger protein 11 by miR-19b-3p resulted in accumulation of nuclear factor kappa B in the nucleus, which in turn led to higher production of inflammatory cytokines.In vivosilencing of miR-19b-3p by a specific antagomir reinvigorates the expression level of RNF11, which in turn reduces the production of inflammatory cytokines, abrogates gliosis and neuronal cell death, and eventually improves the survival rate in the mouse model. Collectively, our results demonstrate that miR-19b-3p positively regulates the JEV-induced inflammatory response. Thus, miR-19b-3p targeting may constitute a thought-provoking approach to rein in JEV-induced inflammation. IMPORTANCE: Japanese encephalitis virus (JEV) is one of the major causes of acute encephalitis in humans worldwide. The pathological features of JEV-induced encephalitis are inflammatory reactions and neurological diseases resulting from glia activation. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally. Accumulating data indicate that miRNAs regulate a variety of cellular processes, including the host inflammatory response under pathological conditions. Recently, a few studies demonstrated the role of miRNAs in a JEV-induced inflammatory response in microglia; however, their role in an astrocyte-derived inflammatory response is largely unknown. The present study reveals that miR-19b-3p targets ring finger protein 11 in glia and promotes inflammatory cytokine production by enhancing nuclear factor kappa B activity in these cells. Moreover, administration of an miR-19b-3p-specific antagomir in JEV-infected mice reduces neuroinflammation and lethality. These findings suggest a new insight into the molecular mechanism of the JEV-induced inflammatory response and provide a possible therapeutic entry point for treating viral encephalitis.

The A66G back mutation in NS2A of JEV SA14-14-2 strain contributes to production of NS1' protein and the secreted NS1' can be used for diagnostic biomarker for virulent virus infection.

Japanese encephalitis virus (JEV) is the most common cause of the prevalent encephalitis in Asia-Pacific region and poses a serious risk to public health. Here, we developed a reliable reverse genetics system based on the JEV SA14-14-2 strain to further explore the mechanism for the synthesis of NS1' protein and to investigate the function of NS1' protein during virus infection. NS1' is an additional form of NS1 protein with 52 amino acid carboxy-terminal extension and is expressed by the members of the Japanese encephalitis (JE) serogroup due to the translation frameshift. A66G substitution in NS2A gene of JEV SA14-14-2 strain contributed to recover the GC-rich pseudoknot and resulted in the formation of the NS1'. The NS1' protein has no significant effect on the virus replication properties in BHK-21 cells. Animal experiments demonstrated that the NS1' protein had a rather minor effect on neurovirulence of JEV SA14-14-2 strain. But the NS1'-expressing virus (rA66G) could induce a higher humoral immune response than the NS1'-non-expressing virus (rSA14-14-2). NS1' protein can be detected in the serum of JEV rA66G infected animal and in the cultural media of that infected mammalian cells. Interesting, only the dimer of NS1' can be detected in the cultural media of the infected BHK-21 cells and the amount of the secreted NS1' was in agreement with that of the secreted virion. In comparison with the live-attenuated JE vaccine strain which is incapable of formation of NS1', most of the virulent JEV strains produce the NS1' protein. And the secreted NS1' may serve as an early surrogate biomarker for viremia to distinguish the field infection from the vaccine inoculation. In total, in the present study, we identified the nt 66 in the viral NS2A gene as one of the critical site for the -1 programmed ribosomal frameshift to produce the NS1' protein and demonstrated the secreted NS1' could be used for diagnostic biomarker during JEV infection.

Toll-like receptor-3 gene polymorphism in patients with Japanese encephalitis.

INTRODUCTION: Japanese encephalitis (JE) is one of the most lethal mosquito-borne viral encephalitis seen in India. Toll-like receptors (TLRs) play a critical role in host defence mechanism against flaviviruses causing encephalitis. We assessed whether abnormalities in toll-like receptor (TLR3) increase the susceptibly for JE. METHOD: A total of 103 JE patients (all from an endemic area) and 103 healthy control subjects were examined for TLR3 Leu412Phe polymorphism with the help of polymerase chain reaction (PCR) and genetic sequencing method. RESULTS: A significantly higher frequency of Leu412Phe polymorphism was noted in JE patients as compared with healthy controls [mutant (TT) genotype, P-value=0.019; mutant (TT)+heterozygous (CT) genotype, P-value=0.013]. Furthermore, frequency of 412Phe allele (T) of TLR3 gene was significantly higher in patients with JE than in controls (P-value=0.001). There was no significant difference in the distribution of any of the TLR3 Leu412Phe (L412F) polymorphism genotype with death within 1month. CONCLUSION: TLR3 gene polymorphism might confer host genetic susceptibility to JE in Indian population. TLR3 polymorphism did not affect the mortality.

Qualitative differences in brain-infiltrating T cells are associated with a fatal outcome in mice infected with Japanese encephalitis virus.

Japanese encephalitis (JE) is the most important form of viral encephalitis in Asia. The critical factors determining mortality and severity of JE virus (JEV) infection remain unclear. We identified brain-infiltrating T cells associated with a fatal outcome of JEV infection in mice. Dying mice were defined as those that lost more than 25 % of their body weight by day 13 and died by day 21, while surviving mice were defined as those that lost less than 10 % by day 13, based on the result of the survival time course study. Two groups of five mice that demonstrated brain virus titers of >1 × 10(6) pfu/g were randomly selected from the dying and surviving groups and used in the analyses. Cytokine patterns in brains were first examined, revealing a higher ratio of Th1-related cytokine genes in dying mice. The expression levels of CD3, CD8, CD25, and CD69 increased in JEV-infected mice relative to mock-infected mice. However, expression levels of these cell-surface markers did not differ between the two groups. T-cell receptor (TCR) usage and complementary determining region 3 (CDR3) sequences were analyzed in the brain-infiltrating T cells. T cells expressing VA8-1, VA10-1, and VB2-1 increased in both groups. However, the dominant T-cell clones as defined by CDR3 amino acid sequence differed between the two groups. The results indicate that the outcome of JEV infection, death or survival, was determined by qualitative differences in infiltrating T-cell clones with unique CDR3 amino acid sequences.