Japanese Encephalitis Virus Infection among Wild Caught Vectors Mosquitoes and Domestic Pigs in Northern West Bengal, India.

BACKGROUND: Japanese encephalitis (JE) is an emerging zoonotic disease caused by JE virus (JEV) and transmitted to humans from pigs or aquatic birds by vector mosquitoes in southeast Asian countries. In this study, JEV infection rate among vector mosquitoes and domestic pigs was determined by detecting viral RNA and anti-JEV antibody (immunoglobulin G), respectively. MATERIALS AND METHODS: A total of 146 pool mosquitoes of Culexvishnui subgroup and 278 pig blood samples were analyzed by reverse transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay methods, respectively. E and premembrane (PrM) gene of JEV detected among vectors were sequenced and a phylogenetic tree was constructed. RESULTS: Five (5.81%) pools of Culextritaeniorhynchus were positive for JEV with pooled infection rate 1.70/1000 mosquitoes. A total of 108 (38.84%) blood samples were positive for anti-JEV antibody. Phylogenetic analysis revealed that our own E and PrM gene sequence of JEV belonging to Genotype III and showed 96.95% sequence similarities with the vaccine strain SA14-14-2. CONCLUSION: It was observed that domestic pigs of northern West Bengal were highly infected with JEV. Hence, the transmission should be blocked by pig vaccination. A pilot study may be undertaken for mass vaccination of the prevailing pig population to observe any reduced rate of JEV transmission from both pig to pig and pig to human.

Differential expression of circulating microRNAs in serum: Potential biomarkers to track Japanese encephalitis virus infection.

Japanese encephalitis is one of the serious vector-borne viral encephalitis diseases found worldwide and poses a major threat to public health. Most Japanese encephalitis virus (JEV) infections are subclinical; only 1: 250 to 1:1000 infected persons develop clinical presentations. Delay in proper diagnosis of JE affects the timeliness of treatment initiation and increases the mortality rate in patients. Therefore, there is an extreme need to develop potential biomarkers, which might improve the diagnosis and can become the basis for development of new therapeutics. The microRNAs (miRNAs/or miRs) are small noncoding RNAs of 17-24 nucleotides that are known to regulate about 60% of human genes. Although miRNAs have been found to regulate various aspects of innate and adaptive immune responses, less information on circulating miRNAs in JE is known. The study of JEV infected human serum miRNAs will provide novel information for the diagnosis of JE as well as for the improvement of disease outcome. Total RNA, including miRNA, was extracted from serum followed by the complementary DNA (cDNA) synthesis by using sequence-specific primers. cDNA was amplified using target-specific TaqMan MicroRNA Assay. Real-time polymerase chain reaction data was normalized using both exogenous (cel-miR-39) and endogenous (hsa-miR-93) controls. We have found significantly altered expression of miR-155 and miR-21 in serum of JEV infected patients as compared to healthy controls, revealing their role as a a noninvasive biomarker in JE. A significant correlation between miRNAs and JE was observed that offers the basis for miRNAs to serve as a new component to develop possible therapeutic strategies for JE in near future.

First report on the detection of Japanese encephalitis virus in fruit bats from India.

Japanese encephalitis (JE) is a mosquito borne viral zoonotic disease and JE virus (JEV) is responsible for causing several children deaths every year in India. Since 1978, cases of JE have been reported from Gorakhpur district of Uttar Pradesh state annually. The knowledge on the role played by wildlife reservoirs in the sylvatic transmission and maintenance of JE virus remains limited. Bats are reservoir hosts for several emerging and re-emerging viral pathogens but their role in zoonotic cycle of JEV has not been elucidated yet. In Gorakhpur district of Uttar Pradesh, 52 fruit bats were found dead on 26 May 2020. The post-mortem report of the bat samples conducted at the Indian Veterinary Research Institute stated that the bats died due to brain hemorrhage, caused by excessive heat. The brain tissue samples of the bats were subjected to investigation using molecular techniques to determine the presence of JEV. The present work reports for the first time the detection of JEV in brain samples of bats from India. The viral load ranging from 8 to 18 copies/reaction was detected in brain samples by TaqMan real Time RT-PCR. The low viral load might be the reason for the absence of apparent clinical signs in bats and suggests the probable role of fruit bats in maintaining the JEV in nature.

Purification of cell-derived Japanese encephalitis virus by dual-mode chromatography.

Purification of the enveloped virus poses a challenge as one must retain viral infectivity to preserve immunogenicity. The traditional process of virus purification is time-consuming, laborious and hard to scale up. Here, a rapid, simple and extensible laboratory program for the purification of Japanese encephalitis virus (JEV) was developed by using differential centrifugation, ultrafiltration, Sepharose 4 fast flow gel chromatography, and CaptoTM Core 700 chromatography. The entire process recovered 61.64% of the original virus, and the purified virus particles maintained good activity and immunogenicity. The purification process described has potential application in large-scale production of high-purity JEV.

Efficient control of Japanese encephalitis virus in the central nervous system of infected pigs occurs in the absence of a pronounced inflammatory immune response.

BACKGROUND: Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia. JEV infection of mice and humans can lead to an uncontrolled inflammatory response in the central nervous system (CNS), resulting in a detrimental outcome. Pigs act as important amplification and reservoir hosts, and JEV infection of pigs is mostly subclinical. Information on virus spread in the CNS and immune responses controlling JEV infection in the CNS of pigs, however remains scarce. METHODS: Nine-week-old pigs were inoculated intranasal or intradermal with a relevant dose of 105 TCID50 of JEV genotype 3 Nakayama strain. Clinical signs were assessed daily, and viral spread was followed by RT-qPCR. mRNA expression profiles were determined to study immune responses in the CNS. RESULTS: Besides a delay of 2 days to reach the peak viremia upon intranasal compared to intradermal inoculation, the overall virus spread via both inoculation routes was highly similar. JEV appearance in lymphoid and visceral organs was in line with a blood-borne JEV dissemination. JEV showed a particular tropism to the CNS but without the induction of neurological signs. JEV entry in the CNS probably occurred via different hematogenous and neuronal pathways, but replication in the brain was mostly efficiently suppressed and associated with a type I IFN-independent activation of OAS1 expression. In the olfactory bulb and thalamus, where JEV replication was not completely controlled by this mechanism, a short but strong induction of chemokine gene expression was detected. An increased IFNy expression was simultaneously observed, probably originating from infiltrating T cells, correlating with a fast suppression of JEV replication. The chemokine response was however not associated with the induction of a strong inflammatory response, nor was an induction of the NLRP3 inflammasome observed. CONCLUSIONS: These findings indicate that an adequate antiviral response and an attenuated inflammatory response contribute to a favorable outcome of JEV infection in pigs and help to explain the limited neurological disease compared to other hosts. We show that the NLRP3 inflammasome, a key mediator of neurologic disease in mice, is not upregulated in pigs, further supporting its important role in JEV infections.

Japanese Encephalitis Virus-Induced Anti-N-Methyl-D-Aspartate Receptor Encephalitis: A Case Report and Review of Literature.

Anti-N-methyl-D-aspartate receptor encephalitis (anti-NMDARe) was originally described as a paraneoplastic disease with more than 50% cases involving a tumor. However, tumor incidence in anti-NMDARe in children is much lower. Herpes simplex virus-induced anti-NMDARe has been well-described; however, findings on Japanese encephalitis virus (JEV)-induced anti-NMDARe are scarce. Here, we describe a 7-year-old boy who presented with fever and headache that progressed to seizures and disturbance of consciousness. Brain magnetic resonance imaging (MRI) revealed abnormalities in the bilateral globus pallidus. The diagnosis of JE was made based on a positive JE antibody test results in serum and cerebrospinal fluid. Antiviral and symptomatic therapies led to rapid recovery. Four weeks after the onset of JE, the patient presented with emotional and behavioral disturbances, sleep difficulty, and extrapyramidal symptoms. MRI showed symmetrical lesions in the bilateral thalami and basal ganglia which were expanded than those on the original scan. Antibodies against NMDAR were detected and immunotherapy led to significant recovery. This case and our literature review suggest that JEV may be a clinically important cause of anti-NMDARe in children. Patients with JE-induced anti-NMDARe present with symptoms similar to those of patients with primary anti-NMDARe. Most patients with JE-induced anti-NMDARe showed a good response to first-line immunotherapies.

Analysis of cross-reactivity between flaviviruses with sera of patients with Japanese encephalitis showed the importance of neutralization tests for the diagnosis of Japanese encephalitis.

Japanese encephalitis (JE) is one of the most important viral encephalitis in Asia. JE is caused by the Japanese encephalitis virus (JEV), which belongs to the genus Flavivirus, family Flaviviridae. The diagnosis of JE is usually based on serological assays, and it has been reported that cross-reactivity between flaviviruses has complicated the interpretations of results from serological assays. Therefore, analysis of the cross-reactivity is an important subject for serological diagnosis of JE and other diseases caused by flaviviruses. In the present study, the cross-reactivity of the sera of patients with JE to other flaviviruses was analyzed using enzyme-linked immunosorbent assay (ELISA) and neutralization tests. Sixteen serum samples were collected from patients with JE and were tested for: i) IgM antibody against West Nile virus (WNV), dengue virus (DENV), zika virus (ZIKV), and tick-borne encephalitis virus (TBEV) using IgM-ELISA, ii) IgG antibody against DENV and TBEV using IgG-ELISA, and iii) neutralization tests with DENV 1-4, ZIKV, TBEV, and WNV. Out of the 16 samples tested using ELISA, 11 and 14 samples were positive for IgM and IgG, respectively, against at least one of the other flaviviruses. In neutralization tests, neutralizing potency against DENV, ZIKV, or TBEV was not detected in any samples. Although 13 samples showed neutralizing potency against WNV, their neutralizing antibody titers were equal to or less than one-eighth of those against JEV. These results show that neutralization tests are more specific than ELISA, indicating the importance of the neutralization tests in the diagnosis of JE.

TaqMan real-time RT-PCR assay for detecting Japanese encephalitis virus in swine blood samples and mosquitoes.

Japanese encephalitis (JE) is an emerging mosquito-borne zoonotic flaviviral disease. The present study was undertaken with the objective to develop TaqMan real-time reverse-transcription polymerase chain reaction (RT-PCR) assay for rapid detection and quantification of Japanese encephalitis virus (JEV) in swine blood and mosquito vectors. The amplification of envelope (E) gene was targeted by designing gene-specific MGB TaqMan fluorescent probe along with the primers. The best performance in terms of sensitivity was achieved by standardized TaqMan real-time RT-PCR with a detection limit of 2.8 copies/reaction and it was found to be 4-log more sensitive than conventional RT-PCR. The applicability of the standardized TaqMan assay was evaluated by screening representative sets of field swine blood samples and mosquito pools for JEV. The viral load ranged between 3.32 × 107-4.2 × 102 copies/ml of swine blood samples, and 5.7 × 109-1.3 × 102 copies/pool of mosquitoes. The standardized assay which is highly sensitive, specific and rapid would aid in screening sentinel swine and mosquitoes under JEV surveillance programs for effective prevention and control of disease in human beings.

[The progress and challenge of Japanese encephalitis control and prevention in China].

Japanese encephalitis (JE) is the important viral encephalitis in China. In the 1940s, JE was confirmed to be epidemic in China. In 1971, the annual incidence rate was 20.92/100 000. Since 2008, JE vaccine was included in the national Expanded Program of Immunization (EPI). In 2013, the incidence of Japanese encephalitis decreased to 0.16/100 000. JE virus is divided into five genotypes, and genotype 1, 3 and 5 JE virus was isolated in China. Genotype 1 JE virus was the mainly genotype currently circulated in China. In recent years, the characteristics of the population of JE have been changed to adult, especially in the northern provinces of China. JE prevention and control faces new challenges.

[Etiologic surveillance and analysis of acute meningitis and encephalitis syndrome in Jinan city in 2013-2016].

Objective: To characterize the etiology and epidemiological characteristics of the acute meningitis and encephalitis syndrome (AMES) in Jinan city in 2013-2016. Methods: The epidemiological data, clinical diagnosis, serum and cerebrospinal fluid (CSF) specimens were collected from 3 577 AMES cases in 6 sentinel hospitals in Jinan city in 2013-2016. Samples of all cases were made sero-diagnosis for Immunoglobulin (Ig) M antibody to Japanese encephalitis virus (JEV) and negative cases of JEV for enterovirus (EV), mumps virus (MuV) and herpes simplex virus (HSV) by enzyme-linked immunosorbent assay (ELISA). Virus isolation and molecular identification were performed. Positive rates were analyzed by Chi-square test. Results: In 2013-2016, the positive rates of JEV, EV, MuV and HSV were 9.0% (322/3 577 cases), 22.1% (643/2 916 cases), 9.9% (289/2 916 cases), 26.9% (783/2 916), respectively. Of these, the positive rates of JEV were 32.9% (261/794), 1.2% (14/1 175), 1.0% (8/807) and 4.9% (39/801 cases); EV: 19.5% (91/466), 35.1% (342/974 cases), 15.5% (115/743) and 13.0% (95/733); MuV: 9.2% (43/466), 14.4% (140/974), 9.0% (67/743) and 5.3% (39/733). HSV: 35.4% (165/466), 38.5% (375/974), 25.7% (191/743) and 7.1% (52/733). There were significant differences in positive rates of 4 kinds of viruses in 2013-2016 (P<0.001). A total of 81 EV strains belonging to 8 serotypes were isolated from 1 020 CSF specimens. The positive rates were 4.8% (6 cases), 13.1% (55 cases), 4.1% (7 cases) and 4.2% (13 cases) from 2013 to 2016. Coxsackievirus (CV) B5, echovirus (E) 6 and E30 accounted for 46% (37 isolates), 22% (18 isolates) and 21% (17 isolates) of all strains. Conclusion: The AMES cases in Jinan city in 2013-2016 were mainly caused by HSV, EV, MuV, JEV. CVB5, E6 and E30 were the dominant serotypes of EV associated with AMES cases in Jinan city.

First co-infection case of melioidosis and Japanese encephalitis in China.

BACKGROUND: Melioidosis is endemic in Southeast Asia and northern Australia. Infection usually follows percutaneous inoculation or inhalation or ingestion of the causative bacterium, Burkholderia pseudomallei, which is present in soil and surface water in endemic regions. Japanese encephalitis (JE) is a vector-borne viral zoonosis caused by Japanese encephalitis virus (JEV), leading to epidemic encephalitis in Southeast Asia. Both B. pseudomallei and JEV have spread dominantly in the Hainan and Guangdong provinces in China. Here we reported the first case of co-infection of B. pseudomallei and JEV, which was discovered in Huizhou in the Guangdong province in June 2016. CASE PRESENTATION: A 52-year-old man was admitted to the hospital with acute febrile illness and headache, diagnosed as respiratory infection, central nervous system (CNS) infection, septicemia, and hepatic dysfunction. Based on B. pseudomallei-positive blood and cerebrospinal fluid (CSF) cultures, the patient was diagnosed with melioidosis and treated aggressively with antibiotics. However, the patient failed to make a full recovery. Further laboratory tests focused on CNS infection were conducted. The co-infection of B. pseudomallei and JEV was confirmed after the positive IgM antibodies of JEV were detected in both CSF and blood. After diagnosis of co-infection with B. pseudomallei and JEV, the patient was provided supportive care in hospital and recovered after approximately 3 weeks. CONCLUSION: Given the possibility of co-infection of B. pseudomallei and JEV, as well as variable case presentations, it is critical to enhance the awareness, detection, and treatment of co-infection in regard to melioidosis.

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.

Changes of Epidemiological Characteristics of Japanese Encephalitis Viral Infection and Birds as a Potential Viral Transmitter in Korea.

Japanese encephalitis (JE) cases have been increasingly reported recently especially in Seoul and its vicinity. Pigs are known as amplifying host of JE virus (JEV), but do not play an important role in these recent events because pig-breeding is not common in Seoul. The distribution and the density of migratory birds are correlated with JE cases in cities and they might be highly potential hosts contributing to transmit JEV in metropolitan areas. JE genotype and sero-prevalence in birds should be determined for the verification of the transmission route of JEV in the recent sporadic occurrence of JE cases in Seoul.

TaqMan Real-time RT-PCR Assay for Detecting and Differentiating Japanese Encephalitis Virus.

OBJECTIVE: To detect Japanese encephalitis virus (JEV) rapidly and distinguish its genotypes, a TaqMan-based reverse transcriptase quantitative polymerase chain reaction (RT-PCR) detection system was developed. METHODS: By aligning the full-length sequences of JEV (G1-G5), six sets of highly specific TaqMan real-time RT-PCR primers and probes were designed based on the highly conserved NS1, NS2, and M genes of JEV, which included one set for non-specific JEV detection and five sets for the detection of specific JEV genotypes. Twenty batches of mosquito samples were used to evaluate our quantitative PCR assay. RESULTS: With the specific assay, no other flavivirus were detected. The lower limits of detection of the system were 1 pfu/mL for JEV titers and 100 RNA copies/µL. The coefficients of variation of this real-time RT-PCR were all < 2.8%. The amplification efficiency of this method was between 90% and 103%. CONCLUSION: A TaqMan real-time RT-PCR detection system was successfully established to detect and differentiate all five JEV genotypes.

Prevalence and risk factors of Japanese encephalitis virus (JEV) in livestock and companion animal in high-risk areas in Malaysia.

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.

Virulence of Japanese Encephalitis Virus Genotypes I and III, Taiwan.

The virulence of genotype I (GI) Japanese encephalitis virus (JEV) is under debate. We investigated differences in the virulence of GI and GIII JEV by calculating asymptomatic ratios based on serologic studies during GI- and GIII-JEV endemic periods. The results suggested equal virulence of GI and GIII JEV among humans.

Epidemiologic Survey of Japanese Encephalitis Virus Infection, Tibet, China, 2015.

We investigated Japanese encephalitis virus (JEV) prevalence in high-altitude regions of Tibet, China, by using standard assays to test mosquitoes, pigs, and humans. Results confirmed that JEV has spread to these areas. Disease prevention and control strategies should be used along with surveillance to limit spread of JEV in high-altitude regions of Tibet.

Isolation and full-genome sequences of Japanese encephalitis virus genotype I strains from Cambodian human patients, mosquitoes and pigs.

Japanese encephalitis remains the most important cause of viral encephalitis in humans in several southeast Asian countries, including Cambodia, causing at least 65 000 cases of encephalitis per year. This vector-borne viral zoonosis - caused by Japanese encephalitis virus (JEV) - is considered to be a rural disease and is transmitted by mosquitoes, with birds and pigs being the natural reservoirs, while humans are accidental hosts. In this study we report the first two JEV isolations in Cambodia from human encephalitis cases from two studies on the aetiology of central nervous system disease, conducted at the two major paediatric hospitals in the country. We also report JEV isolation from Culextritaeniorhynchus mosquitoes and from pig samples collected in two farms, located in peri-urban and rural areas. Out of 11 reverse-transcription polymerase chain reaction-positive original samples, we generated full-genome sequences from 5 JEV isolates. Five additional partial sequences of the JEV NS3 gene from viruses detected in five pigs and one complete coding sequence of the envelope gene of a strain identified in a pig were generated. Phylogenetic analyses revealed that JEV detected in Cambodia belonged to genotype I and clustered in two clades: genotype I-a, mainly comprising strains from Thailand, and genotype I-b, comprising strains from Vietnam that dispersed northwards to China. Finally, in this study, we provide proof that the sequenced JEV strains circulate between pigs, Culex tritaeniorhynchus and humans in the Phnom Penh vicinity.

Viral aetiologies of acute encephalitis in a hospital-based South Asian population.

BACKGROUND: The aetiological spectrum of acute encephalitis shows inter- and intra-geographical variations. We aimed to identify the viruses that cause infectious encephalitis in Sri Lanka, which represents a South Asian population. METHODS: A cross-sectional study was conducted among 99 patients with encephalitis/meningoencephalitis admitted to two tertiary-care hospitals in Colombo. Cerebrospinal fluid and serum were tested for conventional and emerging encephalitogenic viruses. Specific nucleic acid amplification and antibody assays were used to identify viruses. Plaque reduction neutralization test was done to confirm the diagnosis of West Nile virus (WNV). RESULTS: Patients' age ranged from 1 month to 73 years (mean = 24.91; SD = 21.33) with a male:female ratio of 1.75:1. A viral aetiology was identified in only 27.3%. These included dengue virus (40.7%), Japanese encephalitis virus (25.9%), varicella zoster virus, WNV and probable Epstein Barr virus (11.1% each). None were positive for herpes simplex viruses or cytomegalovirus. Screening for bacterial aetiologies was negative for all patients. There were no distinguishable clinical or laboratory findings between the different viral aetiologies. The case fatality rate was 7%, which was higher among patients with an identified viral aetiology. CONCLUSIONS: A viral aetiology was identified in only about a quarter of patients with encephalitis. Dengue virus accounted for the majority.