Prophylactic Administration of Gut Microbiome Metabolites Abrogated Microglial Activation and Subsequent Neuroinflammation in an Experimental Model of Japanese Encephalitis.

Short-chain fatty acids (SCFAs) are gut microbial metabolic derivatives produced during the fermentation of ingested complex carbohydrates. SCFAs have been widely regarded to have a potent anti-inflammatory and neuro-protective role and have implications in several disease conditions, such as, inflammatory bowel disease, type-2 diabetes, and neurodegenerative disorders. Japanese encephalitis virus (JEV), a neurotropic flavivirus, is associated with life threatening neuro-inflammation and neurological sequelae in infected hosts. In this study, we hypothesize that SCFAs have potential in mitigating JEV pathogenesis. Postnatal day 10 BALB/c mice were intraperitoneally injected with either a SCFA mixture (acetate, propionate, and butyrate) or PBS for a period of 7 days, followed by JEV infection. All mice were observed for onset and progression of symptoms. The brain tissue was collected upon reaching terminal illness for further analysis. SCFA-supplemented JEV-infected mice (SCFA + JEV) showed a delayed onset of symptoms, lower hindlimb clasping score, and decreased weight loss and increased survival by 3 days (p < 0.0001) upon infection as opposed to the PBS-treated JEV-infected animals (JEV). Significant downregulation of inflammatory cytokines TNF-α, MCP-1, IL-6, and IFN-Υ in the SCFA + JEV group relative to the JEV-infected control group was observed. Inflammatory mediators, phospho-NF-kB (P-NF-kB) and iba1, showed 2.08 ± 0.1 and 3.132 ± 0.43-fold upregulation in JEV versus 1.19 ± 0.11 and 1.31 ± 0.11-fold in the SCFA + JEV group, respectively. Tissue section analysis exhibited reduced glial activation (JEV group─42 ± 2.15 microglia/ROI; SCFA + JEV group─27.07 ± 1.8 microglia/ROI) in animals that received SCFA supplementation prior to infection as seen from the astrocytic and microglial morphometric analysis. Caspase-3 immunoblotting showed 4.08 ± 1.3-fold upregulation in JEV as compared to 1.03 ± 0.14-fold in the SCFA + JEV group and TUNEL assay showed a reduced cellular death post-JEV infection (JEV-6.4 ± 1.5 cells/ROI and SCFA + JEV-3.7 ± 0.73 cells/ROI). Our study critically contributes to the increasing evidence in support of SCFAs as an anti-inflammatory and neuro-protective agent, we further expand its scope as a potential supplementary intervention in JEV-mediated neuroinflammation.

Efficacy of genotype-matched vaccine against re-emerging genotype V Japanese encephalitis virus.

Japanese encephalitis (JE), caused by the Japanese encephalitis virus (JEV), is a highly threatening disease with no specific treatment. Fortunately, the development of vaccines has enabled effective defense against JE. However, re-emerging genotype V (GV) JEV poses a challenge as current vaccines are genotype III (GIII)-based and provide suboptimal protection. Given the isolation of GV JEVs from Malaysia, China, and the Republic of Korea, there is a concern about the potential for a broader outbreak. Under the hypothesis that a GV-based vaccine is necessary for effective defense against GV JEV, we developed a pentameric recombinant antigen using cholera toxin B as a scaffold and mucosal adjuvant, which was conjugated with the E protein domain III of GV by genetic fusion. This GV-based vaccine antigen induced a more effective immune response in mice against GV JEV isolates compared to GIII-based antigen and efficiently protected animals from lethal challenges. Furthermore, a bivalent vaccine approach, inoculating simultaneously with GIII- and GV-based antigens, showed protective efficacy against both GIII and GV JEVs. This strategy presents a promising avenue for comprehensive protection in regions facing the threat of diverse JEV genotypes, including both prevalent GIII and GI as well as emerging GV strains.

Japanese Encephalitis can be devastating.

Abstract: Japanese encephalitis, caused by the JE virus transmitted by mosquitoes, is the most common type of epidemic encephalitis in Asia. It is endemic in most of South and Southeast Asia, but the number of cases can vary greatly between areas. While many infections do not lead to disease, the symptomatic cases can be very severe and life-threatening. It mainly affects children, whereas adults are generally immune to the disease due to either being infected in childhood or receiving vaccination. However, individuals who are not immune, such as travelers from non-endemic countries, are susceptible to the disease when exposed to the virus for the first time, regardless of age. Without antiviral treatment options, vaccination is the only strategy to establish effective protection against Japanese encephalitis.

Epitope(s) involving amino acids of the fusion loop of Japanese encephalitis virus envelope protein is(are) important to elicit protective immunity.

Dengue vaccine candidates have been shown to improve vaccine safety and efficacy by altering the residues or accessibility of the fusion loop on the virus envelope protein domain II (DIIFL) in an ex vivo animal study. The current study aimed to comprehensively investigate the impact of DIIFL mutations on the antigenicity, immunogenicity, and protective efficacy of Japanese encephalitis virus (JEV) virus-like particles (VLPs) in mice. We found the DIIFL G106K/L107D (KD) and W101G/G106K/L107D (GKD) mutations altered the binding activity of JEV VLP to cross-reactive monoclonal antibodies but had no effect on their ability to elicit total IgG antibodies in mice. However, JEV VLPs with KD or GKD mutations induced significantly less neutralizing antibodies against JEV. Only 46% and 31% of the KD and GKD VLPs-immunized mice survived compared to 100% of the wild-type (WT) VLP-immunized mice after a lethal JEV challenge. In passive protection experiments, naïve mice that received sera from WT VLP-immunized mice exhibited a significantly higher survival rate of 46.7% compared to those receiving sera from KD VLP- and GKD VLP-immunized mice (6.7% and 0%, respectively). This study demonstrated that JEV DIIFL is crucial for eliciting potently neutralizing antibodies and protective immunity against JEV. IMPORTANCE: Introduction of mutations into the fusion loop is one potential strategy for generating safe dengue and Zika vaccines by reducing the risk of severe dengue following subsequent infections, and for constructing live-attenuated vaccine candidates against newly emerging Japanese encephalitis virus (JEV) or Japanese encephalitis (JE) serocomplex virus. The monoclonal antibody studies indicated the fusion loop of JE serocomplex viruses primarily comprised non-neutralizing epitopes. However, the present study demonstrates that the JEV fusion loop plays a critical role in eliciting protective immunity in mice. Modifications to the fusion loop of JE serocomplex viruses might negatively affect vaccine efficacy compared to dengue and zika serocomplex viruses. Further studies are required to assess the impact of mutant fusion loop encoded by commonly used JEV vaccine strains on vaccine efficacy or safety after subsequent dengue virus infection.

Development of a live-attenuated chimeric vaccine against the emerging Usutu virus.

Usutu virus (USUV) is an emerging arthropod-borne flavivirus that has expanded into multiple European countries during the past several decades. USUV infection in human has been linked to severe neurological complications, and no vaccine is now available against USUV. In this work, we develop a live-attenuated chimeric USUV vaccine (termed ChinUSUV) based on the full-length infectious cDNA clone of the licensed Japanese encephalitis virus (JEV) vaccine strain SA14-14-2. In vitro studies demonstrate that ChinUSUV replicates efficiently and maintains its genetic stability. Remarkably, ChinUSUV exhibits a significant attenuation phenotype in multiple mouse models even compared with the licensed JEV vaccine. A single immunization with ChinUSUV elicits potent IgG and neutralizing antibody responses as well as T cell response. Passive transfer of sera from ChinUSUV-immunized mice confers significant protection against lethal homologous challenge in suckling mice. Taken together, our results suggest that ChinUSUV represents a potential USUV vaccine candidate that merits further development.

Seroprevalence of dengue, Japanese encephalitis and Zika among long-term expatriates in Thailand.

BACKGROUND: Travel to Southeast Asia increases the likelihood of acquiring mosquito-borne Flavivirus infections such as dengue (DENV), Japanese encephalitis (JEV) and Zika viruses (ZIKV). Expatriates are long-term travellers who have a higher risk of mosquito-borne illness at their destination country. The purpose of this study was to evaluate the seroprevalence of DENV, JEV and ZIKV infections and the determinants contributing to seropositivity among expatriates living in Thailand. METHODS: A cross-sectional study was performed from December 2017 to February 2020. Expatriates from non-Flavivirus endemic countries were recruited. 5 mL of blood was collected for DENV 1-4, JEV and ZIKV antibody testing by plaque reduction neutralization test (PRNT50). Individuals with vaccination histories or diagnoses for dengue, Japanese encephalitis, yellow fever and tick-borne encephalitis were excluded. RESULTS: Among 254 participants, most participants (83.1%) were male, the mean age was 65 years and the median duration of stay in Thailand was 6 years. Seroprevalence rate of any Flavivirus, non-specific DENV, DENV1-4, JEV and ZIKV were 34.3, 30.7, 20.5, 18.1, 18.9, 10.6, 4.7 and 2.8%, respectively. The presence of neutralizing antibodies against DENV1-4 positively correlates with the duration of stay in Thailand. DENV seropositivity was associated with living in urban areas (aOR 2.75, 95% CI 1.36-5.57). Expatriates were unlikely to have detectable anti-JEV antibodies regardless of time spent in a JEV-endemic area. No risk factors were identified that were significantly associated with JEV or ZIKV seropositivity. Only 48.4% received pre-travel counselling services, while only 18.9% visited a travel medicine specialist. CONCLUSIONS: A high proportion (34.3%) of long-term expatriates living in Thailand were seropositive for flavivirus, mainly from dengue (30.7%). To minimize risk, travel medicine practitioners should provide adequate pre-travel health risk information on mosquito-borne flavivirus infection and offer advice on mosquito bite prevention strategies. Dengue vaccine might be considered in high-risk travellers such as long-term expatriate.

Seroprevalence of Japanese encephalitis virus-specific antibodies in Australia following novel epidemic spread: protocol for a national cross-sectional study.

INTRODUCTION: Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes encephalitis and other morbidity in Southeast Asia. Since February 2022, geographically dispersed JEV human, animal and vector detections occurred on the Australian mainland for the first time. This study will determine the prevalence of JEV-specific antibodies in human blood with a focus on populations at high risk of JEV exposure and determine risk factors associated with JEV seropositivity by location, age, occupation and other factors. METHOD: Samples are collected using two approaches: from routine blood donors (4153 samples), and active collections targeting high-risk populations (convenience sampling). Consent-based sampling for the latter includes a participant questionnaire on demographic, vaccination and exposure data. Samples are tested for JEV-specific total antibody using a defined epitope-blocking ELISA, and total antibody to Australian endemic flaviviruses Murray Valley encephalitis and Kunjin viruses. ANALYSIS: Two analytic approaches will occur: descriptive estimates of seroprevalence and multivariable logistic regression using Bayesian hierarchical models. Descriptive analyses will include unadjusted analysis of raw data with exclusions for JEV-endemic country of birth, travel to JEV-endemic countries, prior JEV-vaccination, and sex-standardised and age-standardised analyses. Multivariable logistic regression will determine which risk factors are associated with JEV seropositivity likely due to recent transmission within Australia and the relative contribution of each factor when accounting for effects within the model. ETHICS: National Mutual Acceptance ethical approval was obtained from the Sydney Children's Hospitals Network Human Research Ethics Committee (HREC). Local approvals were sought in each jurisdiction. Ethical approval was also obtained from the Australian Red Cross Lifeblood HREC. DISSEMINATION: Findings will be communicated to participants and their communities, and human and animal health stakeholders and policy-makers iteratively and after final analyses. Understanding human infection rates will inform procurement and targeted allocation of limited JEV vaccine, and public health strategies and communication campaigns, to at-risk populations.

Epidemiology and risk factors of Japanese encephalitis in Taiwan, 2010-2022.

INTRODUCTION: Taiwan introduced a two-dose inactivated Japanese encephalitis (JE) mouse brain-derived (JE-MB) vaccine into routine childhood immunization in 1968, with booster vaccination implemented in 1974 and 1983. In 2017, JE-MB vaccine was replaced by a two-dose live-attenuated chimeric vaccine (JE-CV). After implementation of JE vaccination programs, JE cases have shifted from children to adults. In this study, we described the JE epidemiology and identify high-risk groups to further inform vaccine policy. METHODOLOGY/PRINCIPAL FINDINGS: We extracted data from Taiwan's notifiable disease surveillance database, vital statistics, and employment statistics from 2010 to 2022. Diagnosis of JE was confirmed by JE seroconversion, a four-fold increase in virus-specific antibodies, a positive JE viral nucleic-acid test, or JE virus isolation. From 2010 to 2022, a total of 313 cases of JE were diagnosed, resulting in an overall incidence rate of 0.10 cases per 100,000 person-years and a mortality rate of 0.006 per 100,000 population per year. Among these patients, 64% were male, and the median age was 51 years (range 0-82). Compared with people born in or after 1976 (vaccinated with four doses of JE-MB vaccine or two doses of JE-CV), those born in or before 1962 (unvaccinated) and those born during 1963-1975 (vaccinated with two or three doses of JE-MB vaccine) had a 4.2-fold (95% confidence interval [CI] 3.0-5.7) and 5.9-fold (95% CI 4.3-8.1) higher risk of JE, respectively. The relative risk of working in agriculture, forestry, fishing, or animal husbandry, compared to other occupations, was 5.0 (95% CI 3.5-7.0). CONCLUSIONS/SIGNIFICANCE: In Taiwan, individuals born before 1976 and those employed in agriculture, forestry, fishing, or animal husbandry had a higher risk of JE. We recommend JE vaccination for people in these high-risk groups who have not been fully vaccinated or have an unknown vaccination history.

Cost-effectiveness analysis of Japanese Encephalitis (JE) vaccination program in Bali Province, Indonesia.

OBJECTIVE: The incidence of Japanese Encephalitis (JE) in Bali Province remains high, and is one among the highest in Indonesia. The Indonesian Government initiated the JE vaccination campaign followed by a JE vaccine introduction program in Bali Province in 2018. The JE vaccination program then has been fully integrated into the provincial routine immunization program since 2019. We conducted a retrospective economic analysis of JE vaccination program in Bali Province, Indonesia; considering multiple vaccination strategies. METHODS: We conducted a cost-effectiveness analysis using a decision analytic model comparing two vaccination strategies with no vaccination from the societal and government perspectives. These vaccination strategies were: (1) JE vaccination campaign and introduction program, and (2) a routine JE vaccination program. We compared costs and outcomes for three hypothetical cohorts of 100,000 children followed from birth to the age of 10 years, with impacts measured throughout the child's life-time. We measured the economic consequences as costs per case, per death, and per disability-adjusted life year (DALY) averted. RESULTS: A routine JE immunization program was the most cost-effective strategy with a cost per DALYs averted of US$ 212.59 and US$ 94.09 from the government and societal perspectives respectively. In contrast, costs per DALYs averted through the JE vaccination campaign and introduction strategy was US$ 1,473.53 and US$ 1,224.20 from the government and societal perspectives respectively. CONCLUSIONS: Both JE vaccination strategies are cost-effective but they are not cost-saving when compared to no immunization program.

The Surveillance of Acute Encephalitis Syndrome in Thailand, 2003-2019: A Perspective for Prevention and Control.

Background: Acute encephalitis syndrome (AES) is an infection of the central nervous system with high case-fatality rates. Japanese encephalitis virus (JEV) is the most common vaccine preventable cause of AES in Asia and part of the Western Pacific. In 2003, the JE vaccine was introduced into Thailand's National Immunization Program and expanded to all provinces. This study reviews data from the national surveillance system on the incidence of AES, including Japanese encephalitis in Thailand to guide surveillance, control, and prevention strategies. Materials and Methods: We collected data on all patients diagnosed with AES and reported to the Bureau of Epidemiology, Ministry of Public Health, Thailand, from 2003 to 2019. Results: A total of 9566 AES patients and 266 death cases were reported during these 17 years. Six hundred and forty-two (6.7%) patients were JE with 16 deaths. The incidence of AES increased from 0.47-0.51-1.36 cases per 100,000 population with a preponderance of cases in adults. CFR reduced from 6.25% - 6.94% in 2003-2005 to 0.78% in 2019. AES cases occurred all year round in all the age groups with a male predilection JE vaccination coverage had reached 83% by 2019. The patients were mainly from the north-eastern region of Thailand. Conclusion: Integrated surveillance regular monitoring, strengthening, and making immunization sustainable is required to improve and maintain progress toward JE control and prevention.

PARP1 inhibition protects mice against Japanese encephalitis virus infection.

Japanese encephalitis (JE) is a vector-borne viral disease that causes acute encephalitis in children. Although vaccines have been developed against the JE virus (JEV), no effective antiviral therapy exists. Our study shows that inhibition of poly(ADP-ribose) polymerase 1 (PARP1), an NAD+-dependent (poly-ADP) ribosyl transferase, protects against JEV infection. Interestingly, PARP1 is critical for JEV pathogenesis in Neuro-2a cells and mice. Small molecular inhibitors of PARP1, olaparib, and 3-aminobenzamide (3-AB) significantly reduce clinical signs and viral load in the serum and brains of mice and improve survival. PARP1 inhibition confers protection against JEV infection by inhibiting autophagy. Mechanistically, upon JEV infection, PARP1 PARylates AKT and negatively affects its phosphorylation. In addition, PARP1 transcriptionally upregulates PTEN, the PIP3 phosphatase, negatively regulating AKT. PARP1-mediated AKT inactivation promotes autophagy and JEV pathogenesis by increasing the FoxO activity. Thus, our findings demonstrate PARP1 as a potential mediator of JEV pathogenesis that can be effectively targeted for treating JE.

Production of Plant-Derived Japanese Encephalitis Virus Multi-Epitope Peptide in Nicotiana benthamiana and Immunological Response in Mice.

The current production of the Japanese encephalitis virus (JEV) vaccine is based on animal cells, where various risk factors for human health should be resolved. This study used a transient expression system to express the chimeric protein composed of antigenic epitopes from the JEV envelope (E) protein in Nicotiana benthamiana. JEV multi-epitope peptide (MEP) sequences fused with FLAG-tag or 6× His-tag at the C- or N-terminus for the purification were introduced into plant expression vectors and used for transient expression. Among the constructs, vector pSK480, which expresses MEP fused with a FLAG-tag at the C-terminus, showed the highest level of expression and yield in purification. Optimization of transient expression procedures further improved the target protein yield. The purified MEP protein was applied to an ICR mouse and successfully induced an antibody against JEV, which demonstrates the potential of the plant-produced JEV MEP as an alternative vaccine candidate.

Incidence of Japanese Encephalitis and Acute Encephalitis Syndrome Hospitalizations in the Medium-Endemic Region in Central India.

BACKGROUND: We estimated the incidence of Japanese encephalitis (JE) and acute encephalitis syndrome (AES) following routine immunization with the live-attenuated SA 14-14-2 JE vaccine. METHODS: We implemented enhanced surveillance of AES and JE hospitalizations in endemic districts in Maharashtra and Telangana States during 2015-2016 and 2018-2020. We estimated incidence and compared differences in the incidence of JE and AES between two states, and vaccinated and unvaccinated districts during two study periods. We also considered secondary data from public health services to understand long-term trends from 2007 to 2020. RESULTS: The annual AES incidence rate of 2.25 cases per 100,000 children in Maharashtra during 2018-2020 was significantly lower than 3.36 cases per 100,000 children during 2015-2016. The six JE-vaccinated districts in Maharashtra had significantly lower incidence rates during 2018-2020 (2.03, 95% CI 1.73-2.37) than in 2015-16 (3.26, 2.86-3.70). In addition, the incidence of both JE and AES in two unvaccinated districts was higher than in the vaccinated districts in Maharashtra. Telangana had a lower incidence of both JE and AES than Maharashtra. The AES incidence rate of 0.95 (0.77-1.17) during 2018-2020 in Telangana was significantly lower than 1.67 (1.41-1.97) during 2015-2016. CONCLUSIONS: The annual incidence rate of Japanese encephalitis was < 1 case per 100,000 children. It indicated accelerated control of Japanese encephalitis after routine immunization. However, the annual incidence of acute encephalitis syndrome was still > 1 case per 100,000 children. It highlights the need for improving surveillance and evaluating the impacts of vaccination.

Adults in Northwest China experienced the largest outbreak of Japanese encephalitis in history 10 years after the Japanese encephalitis vaccine was included in the national immunization program: A retrospective epidemiological study.

Mainland China included Japanese encephalitis (JE) vaccine in the national immunization program in 2008 to control the JE epidemic. However, Gansu province in Western China experienced the largest JE outbreak since 1958 in 2018. We conducted a retrospective epidemiological study to explore the causes of this outbreak. We found that adults aged ≥20 years (especially those in rural areas) were the main JE cases in Gansu Province, with a significant increase in the JE incidence in older adults aged ≥60 years in 2017 and 2018. In addition, JE outbreaks in Gansu Province were mainly located in the southeastern region, while the temperature and precipitation in Gansu Province were gradually increasing in recent years, which made the JE epidemic areas in Gansu Province gradually spread to the western of Gansu Province. We also found that adults aged ≥20 years in Gansu Province had lower JE antibody positivity than children and infants, and the antibody positivity rate decreased with age. In the summer of 2017 and 2018, the density of mosquitoes (mainly the Culex tritaeniorhynchus) in Gansu Province was significantly higher than in other years, and the genotype of JEV was mainly Genotype-G1. Therefore, in the future JE control in Gansu Province, we need to strengthen JE vaccination for adults. Moreover, strengthening mosquito surveillance can provide early warning of JE outbreaks and the spread of epidemic areas in Gansu Province. At the same time, strengthening JE antibody surveillance is also necessary for JE control.

Japanese Encephalitis among Adults: A Review.

Japanese encephalitis (JE) is becoming an increasingly important issue among adults. The reasons for this are multifactorial. During the past decades, new areas of Japanese encephalitis virus (JEV) transmission have occurred in several locations, most notably in a markedly expanded area of Australia during 2021-2022. When JEV enters new areas, cases in adults frequently occur. This is unlike the typical pattern in endemic areas where the burden of disease is in children because most adults are protected through natural immunity following earlier exposure to the virus. Even in endemic areas, JEV has become relatively more important in adults because improved JE control through childhood immunization programs has resulted in a substantial decrease in pediatric JE cases and thus more prominence of adult JE cases. Finally, increases in tourism to JE risk areas have resulted in more exposure of adult travelers, who are usually non-immune, to infection in JE risk areas. In this review we describe the increasing importance of JE in adults in some areas and then consider the comparative clinical presentation and severity of illness among children and adults.

Addressing vaccine-preventable encephalitis in vulnerable populations.

PURPOSE OF REVIEW: Vaccinations have been pivotal in lowering the global disease burden of vaccine-preventable encephalitides, including Japanese encephalitis, tick-borne encephalitis, measles encephalitis, and rabies encephalitis, among others. RECENT FINDINGS: Populations vulnerable to vaccine-preventable infections that may lead to encephalitis include those living in endemic and rural areas, military members, migrants, refugees, international travelers, younger and older persons, pregnant women, the immunocompromised, outdoor, healthcare and laboratory workers, and the homeless. There is scope for improving the availability and distribution of vaccinations, vaccine equity, surveillance of vaccine-preventable encephalitides, and public education and information. SUMMARY: Addressing these gaps in vaccination strategies will allow for improved vaccination coverage and lead to better health outcomes for those most at risk for vaccine-preventable encephalitis.