Antiviral drug research for Japanese encephalitis: an updated review.

Japanese encephalitis (JE) caused by the Japanese encephalitis virus (JEV) is one of Asia's most common viral encephalitis. JEV is a flavivirus, common in rural and sub-urban regions of Asian countries. Although only 1% of JEV-infected individuals develop JE, there is a 20-30% chance of death among these individuals and possible neurological sequelae post-infection. No licensed anti-JE drugs are currently available, despite extensive efforts to develop them. Literature search was performed using databases such as PubMed Central, Google Scholar, Wiley Online Library, etc. using keywords such as Japanese encephalitis virus, antiviral drugs, antiviral drug screening, antiviral drug targets, etc. From around 230 papers/abstracts and research reviews retrieved and reviewed for this study, approximately 180 most relevant and important ones have been cited. Different approaches in drug testing and various antiviral drug targets explored so far have been thoroughly searched from the literature and compiled, besides addressing the future perspectives of the antiviral drug development strategies. Although the development of effective anti-JE drugs is an urgent issue, only supportive care is currently available. Recent advancements in understanding the biology of infection and new drug targets have been promising improvements. Despite hindrances such as the unavailability of a proper drug delivery system or a treatment regimen irrespective of the stage of infection, several promising anti-JE candidate molecules are in different phases of clinical trials. Nonetheless, efficient therapy against JEV is expected to be achieved with drug combinations and a highly targeted drug delivery system soon.

Eco-friendly larvicides from Indian plants: Effectiveness of lavandulyl acetate and bicyclogermacrene on malaria, dengue and Japanese encephalitis mosquito vectors.

Mosquitoes (Diptera: Culicidae) are a key threat for millions of people and animals worldwide, since they act as vectors for devastating pathogens and parasites, including malaria, dengue, Japanese encephalitis, filiariasis and Zika virus. Mosquito young instars are usually targeted using organophosphates, insect growth regulators and microbial agents. Indoor residual spraying and insecticide-treated bed nets are also employed. However, these chemicals have negative effects on human health and the environment and induce resistance in a number of vectors. In this scenario, newer and safer tools have been recently implemented to enhance mosquito control. The concrete potential of screening plant species as sources of metabolites for entomological and parasitological purposes is worthy of attention, as recently elucidated by the Y. Tu's example. Here we investigated the toxicity of Heracleum sprengelianum (Apiaceae) leaf essential oil and its major compounds toward third instar larvae of the malaria vector Anopheles subpictus, the arbovirus vector Aedes albopictus and the Japanese encephalitis vector Culex tritaeniorhynchus. GC-MS analysis showed that EO major components were lavandulyl acetate (17.8%) and bicyclogermacrene (12.9%). The EO was toxic to A. subpictus, A. albopictus, and C. tritaeniorhynchus, with LC50 of 33.4, 37.5 and 40.9µg/ml, respectively. Lavandulyl acetate was more toxic to mosquito larvae if compared to bicyclogermacrene. Their LC50 were 4.17 and 10.3µg/ml for A. subpictus, 4.60 and 11.1µg/ml for A. albopictus, 5.11 and 12.5µg/ml for C. tritaeniorhynchus. Notably, the EO and its major compounds were safer to three non-target mosquito predators, Anisops bouvieri, Diplonychus indicus and Gambusia affinis, with LC50 ranging from 206 to 4219µg/ml. Overall, this study highlights that H. sprengelianum EO is a promising source of eco-friendly larvicides against three important mosquito vectors with moderate toxicity against non-target aquatic organisms.

Green synthesis and characterization of silver nanoparticles fabricated using Anisomeles indica: Mosquitocidal potential against malaria, dengue and Japanese encephalitis vectors.

Mosquitoes (Diptera: Culicidae) represent a key threat for millions of people worldwide, since they act as vectors for devastating parasites and pathogens. In this scenario, eco-friendly control tools against mosquito vectors are a priority. Green synthesis of silver nanoparticles (AgNP) using a cheap, aqueous leaf extract of Anisomeles indica by reduction of Ag(+) ions from silver nitrate solution has been investigated. Bio-reduced AgNP were characterized by UV-visible spectrophotometry, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive spectroscopy (EDX) and X-ray diffraction analysis (XRD). The acute toxicity of A. indica leaf extract and biosynthesized AgNP was evaluated against larvae of the malaria vector Anopheles subpictus, the dengue vector Aedes albopictus and the Japanese encephalitis vector Culex tritaeniorhynchus. Both the A. indica leaf extract and AgNP showed dose dependent larvicidal effect against all tested mosquito species. Compared to the leaf aqueous extract, biosynthesized AgNP showed higher toxicity against An. subpictus, Ae. albopictus, and Cx. tritaeniorhynchus with LC50 values of 31.56, 35.21 and 38.08 µg/mL, respectively. Overall, this study firstly shed light on the mosquitocidal potential of A. indica, a potential bioresource for rapid, cheap and effective AgNP synthesis.

Botanical repellents and pesticides traditionally used against hematophagous invertebrates in Lao People's Democratic Republic: a comparative study of plants used in 66 villages.

Hematophagous parasites such as leeches, ticks, mites, lice, bedbugs, mosquitoes, and myiasis-producing fly larvae are common health problems in Lao People's Democratic Republic. Several arthropod-borne infections, e.g., malaria, dengue fever, and Japanese encephalitis, are endemic there. Effective vector control methods including the use of pesticides, insecticide-treated bed nets, and synthetic and plant-based repellents are important means of control against such invertebrates and the pathogens they may transmit or directly cause. In this study, we documented traditional knowledge on plants that are used to repel or kill hematophagous arthropods, including mosquitoes, bedbugs, human lice, mites and ticks, fly larvae, and blood-sucking leeches. Structured interviews were carried out in 66 villages comprising 17 ethnic groups, covering a range of cultures, throughout Lao People's Democratic Republic. A total of 92 plant species was recorded as traditional repellents (including plants for pesticidal usages) in 123 different plant-ectoparasite combinations. The number and species of plants, and animal taxa repelled (or killed) per plant species differed per region, village, and ethnic group. Traditional use was confirmed in the scientific literature for 74 of these plant species, and for an additional 13 species using literature on closely related species. The use of botanical repellents and pesticides from many plant species is common and widespread in the Lao countryside. In the future, the identification of the active components in certain plants to develop more optimal, inexpensive repellents, insecticides, acaricides, or antileech compounds as alternatives to synthetic repellents/pesticides against blood-feeding insects, ticks, mites, and leeches is warranted.

Japanese encephalitis protein vaccine candidates expressing neutralizing epitope and M.T hsp70 induce virus-specific memory B cells and long-lasting antibodies in swine.

Swine are an important amplifier of Japanese encephalitis (JE) virus in the paradomestic environment. In this study, two JE protein vaccine candidates were evaluated for immunogenicity in swine. Both vaccine plasmids are based on a prokaryotic vector pET-32a(+). One plasmid, designated pET-32a(+)-epitope, encode a cassette consisting of a neutralizing epitope on envelope (E) protein of JE virus, whereas the other plasmid, designated pET-32a(+)-epitope-hsp70, express the fusion protein of the epitope and M.T hsp70. Some differences were detected in the immunogenicity of these two proteins in swine. Swine immunized twice with 2000pmol of the neutralizing epitope or the fusion protein developed neutralizing antibody titers of respectively, 154 and 300, and anti-neutralizing epitope antibody titers of 10(4.25) and 10(6.0) by 3 weeks after the second immunization. In addition, swine immunized with the neutralizing epitope emulsified with adjuvant S206 or with imported mineral oil and Tween-80 induced neutralizing antibody titers of 196 and 244, and anti-neutralizing epitope antibody titers of 10(5.25) or 10(5.6) at the same time point. However, swine administered two doses of a commercial JE vaccine (attenuated virus preparation; JEV SA14-14-2 strain) developed less favorable antibody responses with neutralizing antibody titer 40 and anti-neutralizing epitope antibody titers 10(3.7). The anamnestic response was followed by monitoring titers 1 week after boosting with a viral antigen; swine immunized twice with the fusion protein showed a 177-fold increase in anti-neutralizing epitope titer, indicating a strong recall of the antibody response. The animals maintained detectable levels of anti-neutralizing epitope antibody for at least 105 days after two immunizations, indicating that these four protein antigens are able to stimulate virus-specific memory B cells and long-lasting antibodies at higher levels than is achieved using a current commercial attenuated JEV vaccine. The group immunized with the epitope fused to M.T hsp70 made the strongest proliferation of lymphocytes. Through the assay of the amount of interferon (IFN)-gamma and interleukin (IL)-4 in the serum, swine immunized with the fusion protein increased IFN-gamma in the serum which showed that M.T hsp70 potentiated Th1 immune response.

Novel strategy for treatment of Japanese encephalitis using arctigenin, a plant lignan.

UNLABELLED: OBJECTIVES; To evaluate therapeutic efficacy of arctigenin in an experimental model of Japanese encephalitis (JE). METHODS: Four- to 5-week-old BALB/c mice of either sex were infected intravenously with lethal dose of 3 x 10(5) pfu of Japanese encephalitis virus (JEV). By the 9th day post-infection, all untreated animals succumbed to the infection. Arctigenin was dissolved in DMSO at a concentration of 0.5 mg/mL and stored at 4 degrees C. After one day following virus inoculation, animals were given arctigenin intraperitoneally, twice daily (10 mg/kg of body weight) for next 7 days. RESULTS: Treatment with arctigenin provided complete protection against experimental JE. Arctigenin's neuroprotective effect was associated with marked decreases in: (i) viral load; (ii) active caspase-3 activity; (iii) reactive oxygen species and reactive nitrogen species; (iv) microgliosis and proinflammatory cytokines; (v) levels of stress-associated signalling molecules; and (vi) neuronal death. Furthermore, treatment with arctigenin also improves the behavioural outcome following JE. CONCLUSIONS: In conclusion, our findings provide a novel mechanistic insight into the actions of arctigenin in JE. Results from our in vivo and in vitro experiments clearly indicate that arctigenin reduced (i) viral load and viral replication within the brain, (ii) neuronal death and (iii) secondary inflammation and oxidative stress resulting from microglial activation, thereby suggesting its potential for treating JE. The antiviral, neuroprotective, anti-inflammatory and antioxidative effects of arctigenin successfully reduced the severity of disease induced by JEV.

Single dose of inactivated Japanese encephalitis vaccine with poly(gamma-glutamic acid) nanoparticles provides effective protection from Japanese encephalitis virus.

Japanese encephalitis (JE) is a serious disease caused by the JE virus (JEV), and vaccination is the only way to prevent the diseases. In Japan, the only JE vaccine currently available is an inactivated vaccine that requires multiple doses for effective protection; therefore, an effective single-dose vaccine is needed. However, no report of an effective protocol for a single dose of JE vaccine in animals has been published. Here, we evaluated the efficacy of a single-dose vaccination in mice to which the JE vaccine was given with or without adjuvant. Biodegradable poly(gamma-glutamic acid) nanoparticles (gamma-PGA-NPs) were used as a test adjuvant. Remarkably, a single dose of JE vaccine with gamma-PGA-NPs enhanced the neutralizing antibody titer, and all of the immunized mice survived a normally lethal JEV infection, while only 50% of the mice that received a single dose of JE vaccine without gamma-PGA-NPs survived. The use of aluminum as the adjuvant showed similar levels of enhanced efficacy. These results show that gamma-PGA-NPs are a novel and safe adjuvant for JE vaccine, and that a single dose of JE vaccine with gamma-PGA-NPs provides effective protection from lethal JEV in mice. A similar protocol, in which a single dose of JE vaccine is mixed with gamma-PGA-NPs, may be useful for the immunization of humans.

Chikungunya fever, falciparum malaria, dengue fever, Japanese encephalitis... are we listening to the warning signs for public health in India?

The 2005-epidemic of Chikungunya fever highlights the weaknesses of public health in India. The failure to control mosquitoes, and the illnesses transmitted by them, has resulted in recurrent outbreaks all over the country. This is inevitable given the larger scenario: neglect of the basic requirements of health; poor political support for health; a weak public health capacity; centralised programmes for control based on selective interventions, and poorly-planned development projects which have created conditions ideal for the outbreak of disease. All these issues are concerns for public health ethics and must be addressed to tackle the problems posed by mosquito-borne as well as other communicable diseases.

An hsp70 fusion protein vaccine potentiates the immune response against Japanese encephalitis virus.

To evaluate the possibility of developing an effective subunit vaccine against Japanese encephalitis virus (JEV), mice were intraperitoneally immunized with either a neutralizing epitope (a 27-amino-acid region of the JEV E protein), or with a fusion protein between this region and a Mycobacterium tuberculosis hsp70. Both antigens were heterologously expressed in Escherichia coli as fusion proteins with thioredoxin. The fusion protein antigen elicited a higher titer of anti-thioredoxin-neutralizing epitope antibodies and a stronger proliferation of lymphocytes than did either the neutralizing epitope (irrespective of the presence of mineral oil as an adjuvant), or the conventional JEV SA14-14-2 vaccine. Assays of antibody isotype and IFN-gamma and IL-4 content in post-immunization serum showed that the fusion protein elicited a higher IgG2a titer and higher levels of IFN-gamma suggesting a potentiation of the Th1 immune response. The fusion protein antigen elicited a long-lived immune response, and the antibodies were able to neutralize JEV in vitro more strongly than did those elicited by the JEV SA14-14-2 vaccine. Immunization with the fusion protein generated both humoral and cellular immune responses to JEV, and the fusion protein appeared to be a more efficient protectant than the JEV SA14-14-2 vaccine.

A purified inactivated Japanese encephalitis virus vaccine made in Vero cells.

A second generation, purified, inactivated vaccine (PIV) against Japanese encephalitis (JE) virus was produced and tested in mice where it was found to be highly immunogenic and protective. The JE-PIV was made from an attenuated strain of JE virus propagated in certified Vero cells, purified, and inactivated with formalin. Its manufacture followed current GMP guidelines for the production of biologicals. The manufacturing process was efficient in generating a high yield of virus, essentially free of contaminating host cell proteins and nucleic acids. The PIV was formulated with aluminum hydroxide and administered to mice by subcutaneous inoculation. Vaccinated animals developed high-titered JE virus neutralizing antibodies in a dose dependent fashion after two injections. The vaccine protected mice against morbidity and mortality after challenge with live, virulent, JE virus. Compared with the existing licensed mouse brain-derived vaccine, JE-Vax, the Vero cell-derived JE-PIV was more immunogenic and as effective as preventing encephalitis in mice. The JE-PIV is currently being tested for safety and immunogenicity in volunteers.

Prospects for development of a vaccine against the West Nile virus.

Vaccination provides the ultimate measure for personal protection against West Nile disease. The development of a West Nile vaccine for humans is justified by the uncertainty surrounding the size and frequency of future epidemics. At least two companies (Acambis Inc. and Baxter/immuno) have initiated research and development on human vaccines. West Nile encephalitis has also emerged as a significant problem for the equine industry. One major veterinary vaccine manufacturer (Ft. Dodge) is developing formalin-inactivated and naked DNA vaccines. The advantages and disadvantages of formalin-inactivated whole virion vaccines, Japanese encephalitis vaccine for cross-protection, naked DNA, and live attenuated vaccines are described. A novel technology platform for live, attenuated recombinant vaccines (ChimeriVax) represents a promising approach for rapid development of a West Nile vaccine. This technology uses yellow fever 17D as a live vector for envelope genes of the West Nile virus. Infectious clone technology is used to replace the genes encoding the prM and E structural proteins of yellow fever 17D vaccine virus with the corresponding genes of West Nile virus. The resulting virion has the protein coat of West Nile, containing all antigenic determinants for neutralization and one or more epitopes for cytotoxic T lymphocytes. The genes encoding the nucleocapsid protein, nonstructural proteins, and untranslated terminal regions responsible for replication remain those of the original yellow fever 17D virus. The chimeric virus replicates in the host like yellow fever 17D but immunizes specifically against West Nile virus.

Recombinant, chimaeric live, attenuated vaccine (ChimeriVax) incorporating the envelope genes of Japanese encephalitis (SA14-14-2) virus and the capsid and nonstructural genes of yellow fever (17D) virus is safe, immunogenic and protective in non-human primates.

Yellow fever 17D virus, a safe and effective live, attenuated vaccine, was used as a vector for genes encoding the protective antigenic determinants of a heterologous member of the genus Flavivirus, Japanese encephalitis (JE) virus, the leading cause of acute viral central nervous system infection and death throughout Asia. The viral envelope (prM and E) genes of a full-length cDNA clone of YF 17D virus were replaced with the corresponding genes of JE SA14-14-2, a strain licensed as a live, attenuated vaccine in China. Full-length RNA transcripts of the YF/JE chimaera were used to transfect Vero cells. The progeny virus (named 'ChimeriVax-JE'), was used to define safety after intracerebral (i.c.) inoculation of rhesus monkeys. Monkeys (N = 3) inoculated with a high dose (6.6 log10 pfu) developed a brief viremia, showed no signs of illness, developed high titers of anti-JE neutralizing antibody, and had minimal brain and spinal cord lesion scores according to criteria specified in the WHO monkey neurovirulence test. A control group of 3 monkeys that received a lower dose (4.2 log10 pfu) of commercial YF 17D vaccine had slightly higher lesion scores. To develop a lethal monkey model of JE for vaccine protection tests, we inoculated groups of monkeys i.c. or intranasally (i.n.) with a JE virus strain found to be highly neurovirulent and neuroinvasive for mice. Monkeys inoculated i.c., but not i.n., developed severe encephalitis after an incubation period of 8-13 days. The ChimeriVax-JE virus was passed in a cell line acceptable for human use (diploid fetal rhesus lung) and 4.3 or 5.3 log10 pfu were inoculated into groups of 3 monkeys by the subcutaneous route. All 6 animals developed brief viremias (peak titer < 2.0 log10 pfu/ml) and subsequently had anti-JE but no yellow fever neutralizing antibodies. On day 64, the monkeys were challenged i.c. with 5.5 log10 pfu of virulent JE virus. The immunized animals had no detectable viremia post-challenge, whereas 4 unimmunized controls became viremic. Only 1 of 6 (17%) vaccinated monkeys but 4 of 4 (100%) unvaccinated controls developed encephalitis. Histopathological examination 30 days after challenge confirmed that the protected, immunized animals had no or minimal evidence of encephalitis. These data demonstrated the ability of the ChimeriVax-JE to induce a rapid humoral immune response and to protect against a very severe, direct intracerebral virus challenge. Target areas of neuronal damage and inflammation in monkeys infected IC with wild-type JE, the chimaeric virus and YF 17D were similar, indicating that the histopathological scoring system used for the WHO yellow fever monkey neurovirulence test will be applicable to control testing of chimaeric seed viruses and vaccines.

Protective effect of astragali radix by oral administration against Japanese encephalitis virus infection in mice.

We investigated the protective effect of Astragali Radix (AR) by oral administration against Japanese encephalitis virus (JEV) infection in mice, the pharmacological effects of AR extracts (AE) in different origin, and the chemical composition of the AEs. A protective effect was demonstrated in all four AEs used, however, the effective grade for each one was different. In the control group, an increase of hemagglutination inhibition (HI) antibody titer was observed in all mice surviving 25 d after JEV inoculation. However, the increase of HI antibody titer was not observed in some animals administered an AE. In the control group, the rate of HI antibody positive mice was 90% 3 d after JEV inoculation, while the four groups which received the AE had a 30-60% positive rate. In mice which received the AE, the peritoneal exudate cell (PEC) numbers increased significantly compared to the control group. The predominant cell population of PECs in mice receiving the AE was macrophages, and in the PEC, the active oxygen (AO) production was high. From these results, we propose that the protective effect of AE by oral administration is based on a non-specific mechanism during the early stage of infection, before shifting to antibody production, and that macrophages play an important role in this resistance to JEV infection, e.g., by inducing the production of AO. In the chemical composition of each AE, carbohydrate was the major component.

Protective effect of 6-MFA, a fungal interferon inducer against Japanese encephalitis virus in bonnet macaques.

6-MFA, an extract from the fungus Aspergillus ochraceus was administered to 8 bonnet macaques. An equal number of monkeys matched for age, sex and weight received placebo and served as controls. Twenty hours after the administration of the 6-MFA/placebo the monkeys were challenged with an Indian strain of Japanese encephalitis virus by the intranasal route. Signs and symptoms of the disease such as fever, tremors, loss of appetite, dehydration, flaccid paraplegia or quadriplegia were pronounced in all the control monkeys, while in the 6-MFA treated group only two developed symptoms. Virus could be isolated from only one of the 6-MFA treated monkeys on day 6, and from four control monkeys; one each from CSF, spinal cord, blood and from both nasal swab and blood of the fourth monkey. The appearance of HI and N antibodies in 6-MFA treated group was either delayed or completely suppressed. The results indicate that 6-MFA is a potential antiviral agent which can be used to reduce the morbidity and mortality in bonnet macaques (Macaca radiata) experimentally infected with Japanese encephalitis virus.