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.

Japanese encephalitis: a review of the Indian perspective

Japanese encephalitis virus (JEV) causes Japanese encephalitis, which is a leading form of viral encephalitis in Asia, with around 50,000 cases and 10,000 deaths per year in children below 15 years of age. The JEV has shown a tendency to extend to other geographic regions. Case fatality averages 30% and a high percentage of the survivors are left with permanent neuropsychiatric sequelae. Currently, there is no cure for JEV, and treatment is mainly supportive. Patients are not infectious, but should avoid further mosquito bites. A number of antiviral agents have been investigated; however, none of these have convincingly been shown to improve the outcome of JEV. In this review, the current knowledge of the epidemiology and the pathogenesis of this deadly disease have been summarized.

Preventive strategies for frequent outbreaks of Japanese encephalitis in Northern India.

Japanese encephalitis (JE) remains the most important cause of acute viral encephalitis and continues to spread to hitherto unaffected regions like Indonesia, Pakistan and Australia. Approximately 60% of the world population inhabits JE endemic areas. Despite its restricted range mostly in the developing countries,a high annual incidence of 50,000 cases and about 10,000 deaths has been reported. Disease can be fatal in 25% ases. Magnitude of the problem is even more alarming since the survivors are left with serious long-term neuropsychiatric sequelae. Almost every two years,epidemics of JE occur in Indian subcontinent with a high mortality. JE virus infection results in different disease manifestations in host from mild subclinical febrile illness to clinical infections leading to encephalitis. No antiviral treatment is so far available for JE. The prevention of JE can be achieved by controlling the vector or by immunization regime. The vector control in the rural areas,which are the worst affected ones,is practically almost impossible. Three vaccines that have been implicated against JE include inactivated mouse brain derived, inactivated cell culture derived and cell culture derived live attenuated JE vaccine. But each has its own limitation. Currently,attempts to synthesize recombinant DNA vaccine are being made. New therapeutics are on the way of development like use of minocycline, short interfering RNA, arctigenin, rosmarinic acid, DNAzymes etc. However,the immune mechanisms that lead to JE are complex and need to be elucidated further for the development of therapeutics as well as safe and efficacious JE vaccines.

Sero-survey on Aino, Akabane, Chuzan, bovine ephemeral fever and Japanese encephalitis virus of cattle and swine in Korea

Vector-borne arboviruses produce mild to severe symptoms in domestic animals. Bovine ephemeral fever (BEF), Akabane, Aino, and Chuzan virus have been primarily attributed to reproductive disorders or febrile diseases in cattle, and Japanese encephalitis virus (JEV) is mainly associated with reproductive failures in swine. We investigated antibody titers from domestic swine against four bovine arboviruses (BEF, Akabane, Aino, and Chuzan virus) and from cattle against JEV in Korea. While the positive rates for Akabane and BEF were 37.4% and 15.7%, the positive incidence of Chuzan and Aino were relatively low, with positive rates of 3.04% and 0.4%, respectively, based on a virus neutralization assay. Antibody titers against more than one virus were also frequently detected in domestic swine. The incidence of JEV was 51.3% among domestic cattle. In addition, one positive case was detected in the thoracic fluids from 35 aborted calves, based on the hemagglutination inhibition test. Our results indicate that swine are susceptible hosts of bovine arboviruses without showing clinical symptoms in a natural environment. Moreover, we confirmed that JEV could be associated with reproductive failure in pregnant cattle, as were other vector-borne bovine arboviruses assessed in this study.

Development and evaluation of indirect ELISA for the detection of antibodies against Japanese encephalitis virus in swine

The Japanese encephalitis virus (JEV) is one of causative agents of reproductive failure in pregnant sows. An indirect enzyme-linked immunosorbent assay (I-ELISA) was examined for its potential use in the rapid monitoring of the JEV, and the results were compared with those from the hemagglutination inhibition (HI) and serum neutralization (SN) tests. The comparative analysis showed that the results of I-ELISA showed a significant correlation with the conventional HI (r = 0.867) and SN tests (r = 0.804), respectively. When the I-ELISA results were compared with the traditional diagnostic assays, the sensitivity of the I-ELISA was 94.3% with the HI test and 93.7% with the SN test, respectively. The specificity was found to be 81.4% and 80.0% with the HI and SN tests, respectively. To determine the applicability of I-ELISA in the field, the serum samples from 720 pigs were collected from 4 regions in Korea between July and August 2004. The results indicated that 21.7% of screened pigs were seropositive for the JEV. The seropositive rates of JEV in the 4 provinces were 12.6% in Gyeonggi, 45.0% in Gyeongnam, 16.7% in Jeonbuk, and 12.2% in Jeju. The I-ELISA methodology developed in this study was shown to have considerable sensitivity and specificity through a comparison with HI and the SN tests. Therefore, it might be one of convenient methods for screening a large number of samples in various fields.

First time detection of Japanese encephalitis virus antigen in dry and unpreserved mosquito Culex tritaeniorhynchus Giles, 1901, from Karnal district of Haryana state of India.

Japanese encephalitis virus (JEV) antigen has been detected by antigen capture enzyme linked immunosorbentassay (ELISA) in dry specimens of the mosquito Culex tritaeniorhynchus Giles, 1901, collected from Karnal district of Haryana state in northern India. These mosquitoes were stored in dry condition for 20 months, at room temperature, before processing. The procedure of detecting JEV infection in long time stored, dry vector mosquitoes, has important application in the surveillance of Japanese encephalitis.

A serological survey of arboviral diseases among the human population of the Andaman and Nicobar Islands, India.

In an attempt to determine the prevalence of certain arthropod-borne viruses of public health importance amongst the human population of the Andaman and Nicobar Islands of India, 2,401 sera were collected from six major localities. The sera were analysed by the hemagglutination inhibition (HI) and neutralization (N) tests, using Chikungunya (CHIK), Japanese encephalitis (JE), West Nile (WN), dengue (DEN-2), Langat (TP-21) and Kyasanur Forest disease (KFD) viral antigens. The highest prevalence of HI antibodies was detected against KFD virus (22.4%), followed by Langat (20.2%), JE (5.9%), DEN-2 (3.1%), CHIK (2.9%) and WN (0.8%) viruses. Cross-reactions to the viral antigens were also noted. The results of N tests indicated a high prevalence of DEN-2 (25.4%) virus, followed by Langat (17.5%), CHIK (15.3%), KFD (12%), JE (2.19%) and WN (1.8%). These results are discussed in relation to important epidemiological parameters like age, sex and geographical location. To our knowledge, this is the first report of an extensive serosurvey of arthropod-borne viruses on these islands.

A study on the status of Japanese encephalitis infection in Arunachal Pradesh.

Suspected cases of Japanese Encephalitis (J.E.) were recorded in the hospitals of Arunachal Pradesh from 1986 to 1995. 162 cases were diagnosed as J.E. with predominance in male sex and lower age group. Maximum cases were recorded between June to October. 11.3% pig sera showed J.E. antibodies when tested by ELISA test.

Antigen distribution pattern of Japanese encephalitis virus in Culex tritaeniorhynchus, C. vishnui & C. pseudovishnui.

Distribution of Japanese encephalitis (JE) virus antigen in different tissues of mosquitoes was studied in three species viz., Culex tritaeniorhynchus, C. Vishnui and C. pseudovishnui. Overall per cent positivity was higher in the intrathoracically infected as compared to the orally infected mosquitoes, suggesting the existence of a midgut barrier. The cells at the junction of the foregut-midgut, and midgut-hindgut showed intense fluorescence from the second day post feeding onwards. This suggests that the dissemination of virus takes place from these regions of the gut. A small number of salivary glands were found negative even though fluorescence was seen in the respective head squashes, suggesting involvement of the salivary gland barrier in these mosquitoes. Though there was no difference in the salivary gland positivity between these three species, the salivary gland area positivity was high in C. tritaeniorhynchus mosquitoes. Presence of virus antigen in the ovaries and developing eggs of these three species on the third day suggests the possibility of transovarial transmission of virus even in the first gonotrophic cycle which may have epidemiological importance.

An IgM monoclonal antibody to Japanese encephalitis virus recognizing a cross-reactive epitope on nuclear histones.

An IgM class of monoclonal antibody (MAb) raised against 'envelope' (E) glycoprotein of Japanese encephalitis (JE) virus, cross reacted with nuclear histones, in addition to recognizing the viral antigen present in the cytoplasm of infected cells by indirect fluorescent antibody (FA) technique. The experiments on histone depletion by the acid treatment of uninfected PS (porcine kidney) cells, revealed the loss of nuclear immunofluorescence (IF) which was regained after the reconstitution of acid treated cells with histones, prior-to reacting with MAb NHA-2. The IgM MAb recognized specifically the viral antigens expressed on the surface of JE virus infected PS cells by a modified indirect FA. The adsorption of MAb NHA-2 with calf thymus histones (type II-AS) showed a comparative higher drop in the reactivity to JE virus (54.2% reduction) as compared to that against uncomplexed histones (33.3%) by ELISA, thus indicating a higher MAb affinity to the former. In contrast, the adsorption of MAb with chicken RBC nuclei resulted in comparatively more reduction in the reactivity to the uncomplexed histones (52.4% reduction) as against JE virus (37.5%), suggesting that DNA plays some role in modifying and presenting these epitopes. The cross-linkage of epitopes by glutaraldehyde treatment of JE virus antigen and histones showed a 2-fold and higher rise in the MAb reactivity as against those with unfixed or methanol fixed antigens (no cross-linkage), suggesting that the epitope is conformation dependent. Thus, histones seem to share a partial conformational homology with 'E' glycoprotein of JE virus and immune reaction with histones might lead to an autoimmune disorder.

Prevalence of Japanese encephalitis and West Nile viral infections in pig population in and around Chandigarh.

A study on sero prevalence of Japanese Encephalitis (JE) and West Nile (WN) viruses in pigs was carried out in and around Chandigarh. A total of 158 blood samples collected (June, 1995-March, 1996) from batches of pigs were screened for antibodies against JE and WN viral antigens by haemagglutination inhibition and complement fixation tests. In Chandigarh out of 13 pigs, 5 had JE and one WN HAI antibodies. Similarly, the sera of 14 and 1 pigs in Punjab and 29 and 3 pigs in Haryana were positive for JE and WN HAI antibodies respectively. In total JE specific HAI antibodies were found in 30.3% and CF antibodies in 12.5% of pigs whereas WN specific HAI antibodies were obtained in 3.2% and only one pig had CF antibodies. The results show that JE and WN viral infections are prevalent in pig population in and around Chandigarh.

Detection of HIV seropositivity during an outbreak of Japanese encephalitis in Manipur.

An epidemic outbreak of Japanese encephalitis (JE) occurred during mid 1995. Sixteen serum samples from patients with history of febrile headache, convulsions, mental confusion, neck rigidity etc. were sent to the Department of Virology, School of Tropical Medicine, Calcutta, in August, 1995. Twelve (75%) showed HIV antibody against JEV. Out of these 12 sera showing HIV antibody titre between 1:40 and 1:160, eight (66.6%) showed IgM antibody, giving the presumptive diagnosis of recent JEV infection. Five of these 16 sera showed HIV seropositivity (31.25%). Concomitant JEV and HIV infection could be detected in 3 cases. However, in 2 sera HIV titre were less than 1:20. This is probably the first documentation of concomitant JEV and HIV infection in the eastern India.

Cost benefit analysis of Japanese encephalitis vaccination program in Thailand.

Using decision analysis, we estimated benefits, risks, and costs of implementing the Japanese encephalitis (JE) vaccination program in children aged 18 months and 6 years in Thailand. The costs for inclusion of JE vaccine into the routine immunization program at 18 months and 6 years are $2.16 and $3.68 per person, respectively. In the baseline model, the JE vaccination program will prevent 124 JE cases in the program for 18 months old children and 153 JE cases in the program for 6 years old children. The 18 month child program is more cost-effective than the 6 year child program. The cost-effectiveness ratio in the 18 month child program is $15,715 compared with $21,661 in the 6 year child program. The benefits of the JE vaccination program are the savings in treatment cost, disability care, and the future lifetime earnings from JE prevented. The 18 month child program will save $72,922 per one prevented JE compared with $66,197 in the 6 year child program. The JE vaccination program is cost-beneficial under the base-case assumption. Sensitivity analysis which alters various assumptions indicates that the JE vaccination program is worth implementing unless the incidence of JE is less than 3 per 100,000 population. Otherwise, the cost of vaccine has to be reduced.

An appraisal of some recent diagnostic assays for Japanese encephalitis.

A study was undertaken in South Arcot district of Tamil Nadu, India to assess relative merits of selected diagnostic techniques for Japanese encephalitis. During the transmission seasons of 1993-1995, a total of 85 patients (mostly pediatric) clinically diagnosed as acute encephalitis or other related central nervous system (CNS) disorders were examined; in 53 (62.4%) a laboratory diagnosis of JE was established. In terms of diagnostic value, immunoglobulin M (IgM) antibody capture ELISA (MAC ELISA) on convalescent serum had the highest sensitivity (89%) and negative predictive value (NPV) (50%). This was followed by MAC ELISA on acute serum and CSF which had similar sensitivity (84%) and NPV (40%). The hemagglutination inhibition test and Toxorhynchites splendens inoculation technique for virus isolation were also similar in sensitivity (68%) and NPV (25%). The virus antigen detection technique by IFA in cells of cerebrospinal fluid (CSF) was the least sensitive (58%). The distinct advantage of the acute serum ELISA is that it can be carried out on a single finger-prick blood specimen. The IFA on CSF cells is the most rapid diagnostic test since it requires only 2-3 hours to complete. Therefore, both these tests also offer potential tools for JE surveillance programs.

Evaluation of effectiveness of mouse brain inactivated Japanese encephalitis vaccine produced in India.

Efficacy of mouse brain inactivated Japanese encephalitis vaccine was evaluated by studying the immune status of volunteers 1,2,3,4.5 and 7.5 yr after immunization. Neutralizing (N) antibody which is protective and found to correlate with the immunity after vaccination was estimated in serum by plaque reduction neutralizing test on chick embryo cell monolayer. Mean N-antibody titres of 3.25 (pre-booster) and 3.6, 2.8, 2.06, 1.85 and 1.50 log10 were observed post-booster, and 1,2,3, and 4.5 yr of immunization in volunteers who received complete immunization (3 doses). All the volunteers retained more than 1.0 log10 titre of protective N-antibody in spite of the loss of 0.8, 0.74, 0.21 and 0.35 log10 after 1,2,3, and 4.5 yr respectively. Similarly mean N-antibody titres of 1.6, 3.25, 2.4, 2.25, 1.92 and 1.60 log10 were observed pre-booster, after a single booster dose, and 1,2,3 and 4.5 yr of vaccination in individuals who received only a single booster dose. Ten serum samples of volunteers tested after 7.5 yr of vaccination showed that those who were in constant contact with JE virus (n = 7) in the laboratory maintained high levels of N-antibody whereas others (n = 3) showed a fall in titre indicating the necessity of a booster dose.

Prevalence of hemagglutination-inhibition and neutralizing antibodies to arboviruses in horses of java.

A study was conducted to measure the prevalence of hemagglutination-inhibition (HI) and neutralizing antibodies against two arboviruses (Chikungunya and Japanese encephalitis virus) in horses of Java, Indonesia. Blood specimens were collected from a sample of 112 horses at two stables: Pulo Mas, a racing track-horse complex, located in a residential area in North Jakarta, and Pamulang, a riding school, located in a rural environment of West Jaya. Sera were tested by the HI assay and plaque reduction neutralization test. JEV antibodies were detected by HI in 58 (52%) of the horses, while only 11 (10%) had Chikungunya antibodies by HI. The proportion of Pamulang horses infected with JEV (66%) was significantly higher than found among Pulo Mas horses (40%) screened (p < 0.01). Of the 58 horses with JEV antibodies by HI, 52 (90%) were found to have specific neutralization antibodies to JEV. HI and neutralization tests on horse sera indicated that the risk to alpha virus infections was minimal in horses surveyed from Java. However, there was a high risk of JEV infection among the same population.

The efficacy of Japanese encephalitis vaccine in Henan, China: a case-control study.

A population based case-control study to evaluate Japanese encephalitis (JE) vaccine efficacy was carried out in Gusi County, Henan Province, China from June to September in 1991. This study showed that the JE vaccine had a strong protective effect. The estimate of the vaccine efficacy was 78% (95% CI = 16-94%). An unimmunized child was at 4.54 times greater risk of developing JE than were fully immunized children during the study period. The present study may have underestimated the vaccine efficacy due to evaluation based on routine vaccination which might have been affected by vaccination management and the local cold chain system.