Japanese encephalitis virus (JEV) is an important encephalitis virus in Asia, but there are few data on Malaysia. A hospital-based surveillance system for Japanese encephalitis (JE) has been in operation in Sarawak, Malaysia, for the last 10 years. JEV is endemic in Sarawak, with cases occurring throughout the year and a seasonal peak in the last quarter (one-way anova, P < 0.0001). Ninety-two per cent of 133 cases were children aged 12 years or younger; the introduction of JE vaccination in July 2001 reduced the number of JE cases (84 in the four seasons prior to vs. 49 in the six seasons after, McNemar's test, P = 0.0001). After implementation of the programme, the mean age of infected children increased from 6.3 to 8.0 years (Student's t-test, P = 0.0037), suggesting the need for a catch-up programme.
449 human sera collected in a Land Dyak village were tested for antibodies to 11 arboviruses. Japanese encephalitis and dengue virus antibodies were particularly prevalent. The rates of infection with these viruses were estimated to be 5-2% per annum for Japanese encephalitis, 8-8% for dengue 1 and 4-3% for dengue 2. Chikungunya virus antibodies were quite common with an annual infection rate of the order of 5% per annum. Infections with other Group A and B and Bunyamwera group viruses were generally at a low level.
Japanese encephalitis (JE) is a vector-borne disease caused by the Japanese encephalitis virus (JEV). JEV is transmitted by mosquitoes to a wide range of vertebrate hosts, including birds and mammals. Domestic animals, especially pigs, are generally implicated as reservoirs of the virus, while humans are not part of the natural transmission cycle and cannot pass the virus to other hosts. Although JEV infection is very common in endemic areas (many countries in Asia), less than 1% of people affected develop clinical disease, and severe disease affects about 1 case per 250 JEV infections. Although rare, severe disease can be devastating; among the 30,000-50,000 global cases per year, approximately 20-30% of patients die and 30-50% of survivors develop significant neurological sequelae. JE is a significant public health problem for residents in endemic areas and may constitute a substantial risk for travelers to these areas. The epidemiology of JE and its risk to travelers have changed, and continue to evolve. The rapid economic growth of Asian countries has led to a surge in both inbound and outbound travel, making Asia the second most-visited region in the world after Europe, with 279 million international travelers in 2015. The top destination is China, followed by Thailand, Hong Kong, Malaysia and Japan, and the number of travelers is forecast to reach 535 million by 2030 (+ 4.9% per year). Because of the lack of treatment and the infeasibility of eliminating the vector, vaccination is recognized as the most efficacious means of preventing JE. The IC51 vaccine (IXIARO®) is a purified, inactivated, whole virus vaccine against JE. It is safe, well tolerated, efficacious and can be administered to children, adults and the elderly. The vaccination schedule involves administering 2 doses four weeks apart. For adults, a rapid schedule (0-7 days) is available, which could greatly enhance the feasibility of its use. Healthcare workers should inform both short- and long-term travelers of the risk of JE in each period of the year and recommend vaccination. Indeed, it has been shown that short-term travelers are also at risk, not only in rural environments, but also in cities and coastal towns, especially in tourist localities where excursions to country areas are organized.
Japanese encephalitis virus (JEV) genotype V reemerged in Asia (China) in 2009 after a 57-year hiatus from the continent, thereby emphasizing a need to increase regional surveillance efforts. Genotypic characterization was performed on 19 JEV-positive mosquito pools (18 pools of Culex tritaeniorhynchus and 1 pool of Cx. bitaeniorhynchus) from a total of 64 positive pools collected from geographically different locations throughout the Republic of Korea (ROK) during 2008 and 2010.
The last decade of the 20th Century saw the introduction of an unprecedented number of encephalitic viruses emerge or spread in the Southeast Asian and Western Pacific regions (Mackenzie et al, 2001; Solomon, 2003a). Most of these viruses are zoonotic, either being arthropod-borne viruses or bat-borne viruses. Thus Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has spread through the Indonesian archipelago to Papua New Guinea (PNG) and to the islands of the Torres Strait of northern Australia, to Pakistan, and to new areas in the Indian subcontinent; a strain of tick-borne encephalitis virus (TBEV) was described for the first time in Hokkaido, Japan; and a novel mosquito-borne alphavirus, Me Tri virus, was described from Vietnam. Three novel bat-borne viruses emerged in Australia and Malaysia; two, Hendra and Nipah viruses, represent the first examples of a new genus in the family Paramyxoviridae, the genus Henipaviruses, and the third, Australian bat lyssavirus (ABLV) is new lyssavirus closely related to classical rabies virus. These viruses will form the body of this brief review.
The sharp increase in incidence of dengue infection has necessitated the development of methods for the rapid diagnosis of this deadly disease. Here we report the design and development of a reliable, sensitive, and specific optical immunosensor for the detection of the dengue nonstructural protein 1 (NS1) biomarker in clinical samples obtained during early stages of infection. The present optical NS1 immunosensor comprises a biosensing surface consisting of specific monoclonal NS1 antibody for immunofluorescence-based NS1 antigen determination using fluorescein isothiocyanate (FITC) conjugated to IgG antibody. The linear range of the optical immunosensor was from 15-500ngmL-1, with coefficient of determination (R2) of 0.92, high reproducibility (the relative standard deviation obtained was 2%), good stability for 21days at 4°C, and low detection limit (LOD) at 15ngmL-1. Furthermore, the optical immunosensor was capable of detecting NS1 analytes in plasma specimens from patients infected with the dengue virus, with low cross-reaction with plasma specimens containing the Japanese encephalitis virus (JEV) and Zika virus. No studies have been performed on the reproducibility and cross-reactivity regarding NS1 specificity, which is thus a limitation for optical NS1 immunosensors. In contrast, the present study addressed these limitations carefully where these two important experiments were conducted to showcase the robustness of our newly developed optical-based fluorescence immunosensor, which can be practically used for direct NS1 determination in any untreated clinical sample.
Tick-borne encephalitis virus (TBEV) and Crimean-Congo haemorrhagic fever virus (CCHFV) are important tick-borne viruses. Despite their wide geographical distribution and ease of acquisition, the prevalence of both viruses in Malaysia is still unknown. This study was conducted to determine the seroprevalence for TBEV and CCHFV among Malaysian farm workers as a high-risk group within the population.
The exotic and emerging viral encephalitides are caused by animal or human viruses and characterised by sudden unexpected outbreaks of neurological disease, usually in tropical and sub-tropical regions, but sometimes spreading to temperate areas. Although a wide range of viruses come within this label, as this review highlights, there are common research questions as to the origin and spread of the viruses, the contribution of viral and host factors to the clinical presentations and outcome, and the possibilities for treatment and vaccination.
A dot enzyme immunoassay for determination of antibodies to Japanese encephalitis virus was designed for use as a field technique for the surveillance of Japanese encephalitis virus activity among domestic pigs. The test was compared with the neutralization test and the hemagglutination inhibition test and found to be more sensitive than the hemagglutination inhibition test and comparable to the neutralization test in sensitivity but more simple to perform than either the neutralization or the hemagglutination inhibition tests. An IgM capture ELISA for the determination of JEV specific porcine IgM was also utilized to determine current infection rates in pigs. The tests which do not involve the determination of specific IgM are better used for testing sentinel animals for providing clues as to the rate of transmission of JEV among pigs. IgM tests determining acute infection are less likely to be useful unless animals are tested very frequently or if a great number of animals are tested at any one time.
Dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV) are mosquito-borne flavivirus of medical importance in tropical countries such as Malaysia. However, much remains unknown regarding their prevalence among the underserved indigenous people (Orang Asli) living in communities in the forest fringe areas of Peninsular Malaysia. Information on the prevalence of diseases is necessary to elevate the effectiveness of disease control and preventive measures. This study aimed to determine the seroprevalence of the three major flaviviruses among the Orang Asli and investigate the association between demographic factors and seropositivities. Sampling activities were conducted in the Orang Asli villages to obtain serum samples and demographic data from consenting volunteers. The presence of DENV, JEV, and ZIKV immunoglobulin G (IgG) antibodies in the sera were examined using commercial enzyme-linked immunosorbent assay kits. A focus reduction neutralization assay was performed to measure virus-specific neutralizing antibodies. A total of 872 serum samples were obtained from the Orang Asli volunteers. Serological assay results revealed that DENV IgG, JEV IgG, and ZIKV IgG seropositivities among the Orang Asli were at 4.9%, 48.4%, and 13.2%, respectively. Neutralizing antibodies (FRNT50 ≥ 1:40) against JEV and ZIKV were found in 86.7% and 100.0%, respectively, out of the samples tested. Positive serology to all three viruses corresponded significantly to the age of the volunteers with increasing seropositivity in older volunteers. Findings from the study suggest that Orang Asli are at significant risk of contracting JEV and ZIKV infections despite the lack of active transmission of the viruses in the country.
Japanese encephalitis virus (JEV) infection alters microRNA (miRNA) expression in the central nervous system (CNS). However, the mechanism contributing to miRNA regulation in the CNS is not known. We discovered global degradation of mature miRNA in mouse brains and neuroblastoma (NA) cells after JEV infection. Integrative analysis of miRNAs and mRNAs suggested that several significantly downregulated miRNAs and their targeted mRNAs were clustered into an inflammation pathway. Transfection with miRNA 466d-3p (miR-466d-3p) decreased interleukin-1β (IL-1β) expression and inhibited JEV replication in NA cells. However, miR-466d-3p expression increased after JEV infection in the presence of cycloheximide, indicating that viral protein expression reduced miR-466d-3p expression. We generated all the JEV coding proteins and demonstrated NS3 helicase protein to be a potent miRNA suppressor. The NS3 proteins of Zika virus, West Nile virus, and dengue virus serotype 1 (DENV-1) and DENV-2 also decreased miR-466d-3p expression. Results from helicase-blocking assays and in vitro unwinding assays demonstrated that NS3 could unwind pre-miR-466d and induce miRNA dysfunction. Computational models and an RNA immunoprecipitation assay revealed arginine-rich domains of NS3 to be crucial for pre-miRNA binding and degradation of host miRNAs. Importantly, site-directed mutagenesis of conserved residues in NS3 revealed that R226G and R202W reduced the binding affinity and degradation of pre-miR-466d. These results expand the function of flavivirus helicases beyond unwinding duplex RNA to degrade pre-miRNAs. Hence, we revealed a new mechanism for NS3 in regulating miRNA pathways and promoting neuroinflammation.IMPORTANCE Host miRNAs have been reported to regulate JEV-induced inflammation in the CNS. We found that JEV infection could reduce expression of host miRNA. The helicase region of the NS3 protein bound specifically to miRNA precursors and could lead to incorrect unwinding of miRNA precursors, thereby reducing the expression of mature miRNAs. This observation led to two major findings. First, our results suggested that JEV NS3 protein induced miR-466d-3p degradation, which promoted IL-1β expression and JEV replication. Second, arginine molecules on NS3 were the main miRNA-binding sites, because we demonstrated that miRNA degradation was abolished if arginines at R226 and R202 were mutated. Our study provides new insights into the molecular mechanism of JEV and reveals several amino acid sites that could be mutated for a JEV vaccine.
Japanese encephalitis (JE) is vector-borne zoonotic disease which causes encephalitis in humans and horses. Clinical signs for Japanese encephalitis virus (JEV) infection are not clearly evident in the majority of affected animals. In Malaysia, information on the prevalence of JEV infection has not been established. Thus, a cross-sectional study was conducted during two periods, December 2015 to January 2016 and March to August in 2016, to determine the prevalence and risk factors in JEV infections among animals and birds in Peninsular Malaysia. Serum samples were harvested from the 416 samples which were collected from the dogs, cats, water birds, village chicken, jungle fowls, long-tailed macaques, domestic pigs, and cattle in the states of Selangor, Perak, Perlis, Kelantan, and Pahang. The serum samples were screened for JEV antibodies by commercial IgG ELISA kits. A questionnaire was also distributed to obtain information on the animals, birds, and the environmental factors of sampling areas. The results showed that dogs had the highest seropositive rate of 80% (95% CI: ± 11.69) followed by pigs at 44.4% (95% CI: ± 1.715), cattle at 32.2% (95% CI: ± 1.058), birds at 28.9% (95% CI: ± 5.757), cats at 15.6% (95% CI: ± 7.38), and monkeys at 14.3% (95% CI: ± 1.882). The study also showed that JEV seropositivity was high in young animals and in areas where mosquito vectors and migrating birds were prevalent.
Flaviviruses (FVs) are arthropod-borne viruses of medical and veterinary importance. Numerous species of FVs have been isolated from various host; mainly humans, animals, ticks, and mosquitoes. Certain FVs are extremely host-specific; at the same time, some FVs can infect an extensive range of species. Based on published literatures, 11 species of FVs have been detected from diverse host species in Malaysia. In humans, dengue virus and Japanese encephalitis virus have been reported since 1901 and 1942. In animals, the Batu Cave virus, Sitiawan virus, Carey Island, Tembusu virus, Duck Tembusu virus, and Japanese encephalitis viruses were isolated from various species. In mosquitoes, Japanese encephalitis virus and Kunjin virus were isolated from Culex spp., while Zika virus and Jugra virus were isolated from Aedes spp. In ticks, the Langat virus was isolated from Ixodes spp. One of the major challenges in the diagnosis of FVs is the presence of sero-complexes as a result of cross-reactivity with one or more FV species. Subsequently, the distribution of specific FVs among humans and animals in a specific population is problematic to assess and often require comprehensive and thorough analyses. Molecular assays such as quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and digital droplet RT-PCR (ddRT-PCR) have been used for the differentiation of flavivirus infections to increase the accuracy of epidemiological data for disease surveillance, monitoring, and control. In situations where sero-complexes are common in FVs, even sensitive assays such as qRT-pCR can produce false positive results. In this write up, an overview of the various FV sero-complexes reported in Malaysia to date and the challenges faced in diagnosis of FV infections are presented.
Introduction: Japanese encephalitis (JE) is a vector-borne zoonotic disease caused by the Japanese encephalitis virus (JEV). Paddy fields are high risk area for JEV transmission due the presence of Culex tritaeniorhynchus as vector and wild boar as an amplifier host. Methods: We presented case reports for three patients infected with JEV residing in paddy field area from 2015 to 2018 in Kerian, Perak; illustrating the symptoms and epidemiology trend of the dis-ease. Results: All three cases live next to paddy field and were exposed during peak biting hours. They presented with fever and altered consciousness. IV Acylovir was started as one of the treatments and all had lumbar puncture for CSF testing. Two cases (adult) were confirmed JE after they had passed away. Two cases occurred during fasting month where food wastes were abundance. The source of infection was probably from wild boars that came out at night to hunt for food. Presence of C. tritaeniorhynchus was demonstrated as the main vector in these areas. Conclusion: JE has no cure and human vaccination is the main prevention method. At the moment, vector control, environmental management, chemical control and personal protection are the main mitigation strategies for this area. Further study on JEV and wild board in Kerian will be most beneficial.
Japanese encephalitis virus (JEV) is a representative virus of the JEV serogroup in genus Flavivirus, family Flaviviridae. JEV is a mosquito-borne virus that causes Japanese encephalitis (JE), one of the most severe viral encephalitis diseases in the world. JEV is divided into five genotypes (G1-G5), and each genotype has its own distribution pattern. However, the distribution of different JEV genotypes has changed markedly in recent years. JEV G1 has replaced G3 as the dominant genotype in the traditional epidemic areas in Asia, while G3 has spread from Asia to Europe and Africa and caused domestic JE cases in Africa. G2 and G5, which were endemic in Malaysia, exhibited great geographical changes as well. G2 migrated southward and led to prevalence of JE in Australia, while G5 emerged in China and South Korea after decades of silence. Along with these changes, JE occurred in some non-traditional epidemic regions as an emerging infectious disease. The regional changes in JEV pose a great threat to human health, leading to huge disease burdens. Therefore, it is of great importance to strengthen the monitoring of JEV as well as virus genotypes, especially in non-traditional epidemic areas.
Japanese encephalitis virus (JEV), a member of the family Flaviviridae, causes severe neurological disorders in humans. JEV infections represent one of the most widely spread mosquito-borne diseases, and therefore, it has been considered as an endemic disease. An effective antiviral drug is still unavailable to treat JEV, and current drugs only provide supportive treatment to alleviate the symptoms and stabilize patients' conditions. This study was designed to evaluate the antiviral activity of the sulphated polysaccharides "Carrageenan," a linear sulphated polysaccharide that is extracted from red edible seaweeds against JEV replication in vitro. Viral inactivation, attachment, and post-infection assays were used to determine the mode of inhibition of Carrageenan. Virus titters after each application were evaluated by plaque formation assay. MTT assay was used to determine the 50% cytotoxic concentration (CC50), and ELISA-like cell-based assay and immunostaining and immunostaining techniques were used to evaluate the 50% effective concentration (EC50). This study showed that Carrageenan inhibited JEV at an EC50 of 15 µg/mL in a dose-dependent manner with CC50 more than 200 µg/mL in healthy human liver cells (WRL68). The mode of inhibition assay showed that the antiviral effects of Carrageenan are mainly due to their ability to inhibit the early stages of virus infection such as the viral attachment and the cellular entry stages. Our investigation showed that Carrageenan could be considered as a potent antiviral agent to JEV infection. Further experimental and clinical studies are needed to investigate the potential applications of Carrageenan for clinical intervention against JEV infection.
West Nile virus (WNV) is a zoonotic mosquito-borne flavivirus that is harbored and amplified by wild birds via the enzootic transmission cycle. Wide range of hosts are found to be susceptible to WNV infection including mammals, amphibians and reptiles across the world. Several studies have demonstrated that WNV was present in the Malaysian Orang Asli and captive birds. However, no data are available on the WNV prevalence in wild birds found in Malaysia. Therefore this study was conducted to determine the serological and molecular prevalence of WNV in wild birds in selected areas in the West Coast of Peninsular Malaysia. Two types of wild birds were screened, namely migratory and resident birds in order to explore any possibility of WNV transmission from the migratory birds to the resident birds. Thus, a cross-sectional study was conducted at the migratory birds sanctuary located in Kuala Gula, Perak and Kapar, Selangor by catching 163 migratory birds, and 97 resident birds from Kuala Gula and Parit Buntar, Perak at different time between 2016 and 2017 (Total, n = 260). Blood and oropharyngeal swabs were collected for serological and molecular analysis, respectively. Serum were screened for WNV antibodies using a commercial competitive ELISA (c-ELISA) (ID Screen® West Nile Competition Multi-species ELISA, ID VET, Montpellier, France) and cross-reactivity towards Japanese Encephalitis virus (JEV) was also carried out using the JEV-double antigen sandwich (DAS) ELISA. Oropharyngeal swabs were subjected to one-step RT-PCR to detect WNV RNA, in which positive reactions were subsequently sequenced. WNV seropositive rate of 18.71% (29/155) at 95% CI (0.131 to 0.260) and molecular prevalence of 15.2% (16/105) at 95% CI (0.092 to 0.239) were demonstrated in migratory and resident wild birds found in West Coast Malaysia. Phylogenetic analyses of the 16 WNV isolates found in this study revealed that the local strains have 99% similarity to the strains from South Africa and were clustered under lineage 2. Evidence of WNV infection in resident and migratory birds were demonstrated in this study. As a summary, intervention between migratory birds, resident birds and mosquitoes might cause the introduction and maintenance of WNV in Malaysia, however the assumption could be further proven by studying the infection dynamics in the mosquitoes present in the studied areas.
Duck Tembusu virus (DTMUV), a newly identified flavivirus, has rapidly spread to China, Malaysia and Thailand. The potential threats to public health have been well-highlighted; however its virulence and pathogenesis remain largely unknown. Here, by using reverse genetics, a recombinant chimeric DTMUV based on Japanese encephalitis live vaccine strain SA14-14-2 was obtained by substituting the corresponding prM and E genes (named ChinDTMUV). In vitro characterization demonstrated that ChinDTMUV replicated efficiently in mammalian cells with small-plaque phenotype in comparison with its parental viruses. Mouse tests showed ChinDTMUV exhibited avirulent phenotype in terms of neuroinvasiveness, while it retained neurovirulence from its parental virus DTMUV. Furthermore, immunization with ChinDTMUV was evidenced to elicit robust IgG and neutralizing antibody responses in mice. Overall, we successfully developed a viable chimeric DTMUV, and these results provide a useful platform for further investigation of the pathogenesis of DTMUV and development of a live attenuated DTMUV vaccine candidate.
We have developed and characterised a mouse model of Japanese encephalitis virus (JEV) infection via
footpad inoculation in order to better mimic viral transmission by mosquito bites. Two-week-old and
5-week-old mice consistently developed signs of infection such as ruffled fur, weight loss, hunchback
posture, tremors, mask-like facies and occasionally, hindlimb paralysis at 4 days post infection (dpi)
and 11-13 dpi, respectively. Most of the animals died within 24 to 48 hours following the onset of signs
of infection, with mortalities of 100% and 33.3% in 2-week-old and 5-week-old mice, respectively.
Mild meningitis and variable parenchymal inflammation with formation of microglial nodules, focal
necrosis and neuronophagia, and perivascular cuffing by inflammatory cells were observed in the
caudate nucleus, putamen, thalamus, cerebral cortex, brainstem, and spinal cord. Viral antigens/RNA
were demonstrated by immunohistochemisty and in situ hybridization, respectively, in most of these
areas as well as in the hippocampus and cerebellum, albeit more focally. The pathological findings in
this mouse model were generally similar to human Japanese encephalitis (JE) and other established JE
models but perhaps, compared to other JEV mouse models, it demonstrates lethal encephalitic infection
more consistently. We believe that our mouse model should be useful to study the pathogenesis of JE,
and for testing anti-viral drugs and vaccines
Japanese encephalitis (JE) is a neurotropic flavivirus that causes inflammation in central nervous system (CNS), neuronal death and also compromises the structural and functional integrity of the blood-brain barrier (BBB). The aim of this study was to evaluate the BBB disruption and apoptotic process in Japanese encephalitis virus (JEV)-infected transfected human brain microvascular endothelial cells (THBMECs). THBMECs were overlaid by JEV with different MOIs (0.5, 1.0, 5.0 and 10.0) and monitored by electrical cell-substrate impedance sensing (ECIS) in a real-time manner in order to observe the barrier function of THBMECs. Additionally, the level of 43 apoptotic proteins was quantified in the virally infected cells with different MOIs at 24h post infection. Infection of THBMEC with JEV induced an acute reduction in transendothelial electrical resistance (TEER) after viral infection. Also, significant up-regulation of Bax, BID, Fas and Fasl and down-regulation of IGFBP-2, BID, p27 and p53 were observed in JEV infected THBMECs with 0.5 and 10 MOIs compared to uninfected cells. Hence, the permeability of THBMECs is compromised during the JEV infection. In addition high viral load of the virus has the potential to subvert the host cell apoptosis to optimize the course of viral infection through deactivation of pro-apoptotic proteins.