Japanese Encephalitis: A Neglected Viral Disease and Its Impact on Global Health

Japanese encephalitis (JE) is among the most significant viral encephalitis in Asia, particularly in rural and suburban areas where rice culture and pig farming coexist. It has also occurred rarely and occasionally in northern Australia and some parts of the western pacific. JE is caused due to infection with the JE virus (JEV), a mosquito borne flavivrus. The main JEV transmission cycle involves Culex tritaeniorhynchus mosquitoes and similar species that lay eggs in rice paddies and other open water resources, with pigs and aquatic birds as principal vertebrate amplifying hosts [Han et al., 2012]. Humans are generally considered as dead-end JEV hosts i.e. they rarely develop enough viremia to infect feeding mosquitoes. Nearly 20-30% of JE cases are fatal and 30-50% of survivors have major neurological disorders [Bhattacharyya et al., 2013]. JE is mostly a disease of children but other age groups may be affected [Kundu et al., 2013; Griffiths et al., 2013; Larena et al., 2013]. In most temperate areas of Asia, JEV is transmitted mainly during summer season, when large epidemics can occur. In the tropics and subtropics region, transmission can occur throughout the year but often increases during the rainy season [Campbell et al., 2011]. The first epidemic of JE was recorded in Japan in 1871. Major outbursts have been seen in nearly every 10 years. In 1924 more than 6,000 cases were reported in a major outbreak in Japan [Solomon et al., 2000]. The disorders caused by JEV began from Southeast Asia and now it’s affecting people worldwide [Liu et al., 2013; Li et al., 2013]. Nearly 30 million people are at danger of JEV infection [Saxena et al., 2003]. Though intensive care and support are able to lower the death rate but patients continue to suffer from this disease for a long period of time. Some effects such as learning difficulties and behavioral problems can remain masked for several years.


Introduction
Japanese encephalitis (JE) is among the most significant viral encephalitis in Asia, particularly in rural and suburban areas where rice culture and pig farming coexist. It has also occurred rarely and occasionally in northern Australia and some parts of the western pacific. JE is caused due to infection with the JE virus (JEV), a mosquito borne flavivrus. The main JEV transmission cycle involves Culex tritaeniorhynchus mosquitoes and similar species that lay eggs in rice paddies and other open water resources, with pigs and aquatic birds as principal vertebrate amplifying hosts [Han et al., 2012]. Humans are generally considered as dead-end JEV hosts i.e. they rarely develop enough viremia to infect feeding mosquitoes. Nearly 20-30% of JE cases are fatal and 30-50% of survivors have major neurological disorders [Bhattacharyya et al., 2013]. JE is mostly a disease of children but other age groups may be affected [Kundu et al., 2013;Griffiths et al., 2013;Larena et al., 2013]. In most temperate areas of Asia, JEV is transmitted mainly during summer season, when large epidemics can occur. In the tropics and subtropics region, transmission can occur throughout the year but often increases during the rainy season [Campbell et al., 2011]. The first epidemic of JE was recorded in Japan in 1871. Major outbursts have been seen in nearly every 10 years. In 1924 more than 6,000 cases were reported in a major outbreak in Japan [Solomon et al., 2000]. The disorders caused by JEV began from Southeast Asia and now it's affecting people worldwide . Nearly 30 million people are at danger of JEV infection [Saxena et al., 2003]. Though intensive care and support are able to lower the death rate but patients continue to suffer from this disease for a long period of time. Some effects such as learning difficulties and behavioral problems can remain masked for several years.

Viral replication and morphogenesis
JEV virion comprises of a single strand of positive-sense RNA of around 11kb, enclosed in a nucleocapsid and surrounded by envelope made up of glycoproteins Ye et al., 2012]. The RNA consists of a short 5' untranslated region (UTR), a longer 3' UTR and a single open reading frame between them. It encodes 3432 aminoacid polyprotein, which is translationally and post translationally cleaved by viral and host proteases into three structural proteins (core-C, pre-membrane-PrM and envelope-E) and seven nonstructural (NS) proteins (NS1,NS2A,NS2B,NS3,NS4A,NS4B and NS5) [  The C protein of 12-14 kDa in size is highly basic and fuses with the RNA to form the nucleocapsid. The PrM is in close proximity with the E protein, forming a heterodimer and is believed to act as a 'chaperone' to it, hindering its function until after virion release. Just before the virion release, the PrM protein is cleaved by a protease to its mature M protein form. This alteration contributes for the formation and activation of E protein homodimers. Researchers suggested that the extremely conserved N glycosylation motif N 15 -X 16 -T 17 in JEV PrM and its N-glycan substituents are essential for several stages of JEV biology: PrM biogenesis, virus release and pathogenesis. The E protein is the largest structural protein, comprising of approximately 500 aminoacids, with up to two potential glycosylation sites. It is the main target for the humoral immune response and is believed to play a vital role in viral entry into host cells [Solomon et al., 2003]. The xlink protein is involved in virus replication and regulation of the innate immune response Zhang et al., 2012]. The functions of NS3 and NS4 are prominent, they code for serine protease and RNA dependent RNA polymerase (RdRp) . There is a high rate of mutation in JEV because RdRp is expected to have some error which leads to vast alterations in genomic sequences of JEV worldwide [Saxena et al., 2008]. Since all flaviviral NS proteins are essential for viral replication, any one of them can be selected as target for selective inhibitors of viral replication for therapeutic intervention [Anantpadma et al., 2011;Mastrangelo et al., 2012].

Pathogenesis
The development of JEV infection, beginning from its entry till reaching its site of action, the central nervous system (CNS) is not well understood. Studies with other flaviviruses have brought us to the belief that upon entry through mosquito bite, the virus infects dendritic cells in the skin and is carried to the nearest draining lymph nodes, thereby initiating a round of early immune response. But this response is not enough to counter the virus. Meanwhile the virus spreads to secondary lymphoid organs before entering the blood circulation through the efferent lymphatic system. During the subsequent transient viremia, peripheral organs such as kidney, liver and spleen are known to be infected first, after which the neurotropic virus spreads to the CNS. It is still not clear how JEV is able to escape the host's peripheral immune response. After the virus escapes the immune system, it crosses the Blood Brain Barrier to enter the CNS. JEV may cross the BBB by passive transport across the endothelium, by active replication in endothelial cells or by a 'Trojan Horse' mechanism in which the virus is carried into the brain by infected inflammatory cells. Monocytes and macrophages are considered to be the feasible carriers of the virus in the CNS as the virus can survive for a prolonged time and effectively replicate within these cells. During the entry of infected monocytes and macrophages through the BBB, change in the structural and functional integrity of the BBB, leads to production of matrix metalloproteases released by endothelial cells of BBB. This further result in deterioration of BBB stability. BBB stability. Due to the compromised functioning of the BBB, peripheral inflammatory cells are recruited to the infected brain that extends the neuronal impairment.
JEV causes neuronal damage in the brain. However in several cases, JEV is possibly not directly involved in the destruction of brain tissue but it may activate microglia and trigger cellmediated immune response. Microglial cells are the resident immune cells of the CNS and have a crucial role in host defense against invading microorganisms. Microglial activation is considered as an adaptive response whereby microglia release neuroprotective factors to ease the recovery of injured neurons. They also perform phagocytosis for dying or damaged neurons, before they lyse and release toxic agents into surrounding areas. JEV infection activates microglia both morphologically and functionally, in vivo, which causes rise in the level of pro-inflammatory mediators, such as IL-6, TNF-α, RANTES and MCP-1 [Thongtan et al., 2012]. These proinflammatory mediators and cytotoxins released from activated microglia are involved in inducing neuronal death that complements JE. Neuronal death by secreted TNF is mediated by the TNF receptor-associated death domain protein (TRADD), which there upon regulates a downstream apoptotic cascade, in neurons [Swarup et al., 2007]. During JEV infection, nitric oxide (NO) is released by macrophages and plays a significant role in inflammation, even though NO itself is a strong antimicrobial agent, researchers have suggested that it strongly inhibits synthesis of viral RNA, protein accumulation and virus release from infected cells. NO production is higher in the JEV infected brain, and plays a crucial role in the innate immunity of the host and its ability to restrict the initial stages of JEV infection in the CNS [Saxena et al., 2000].
In addition to neuronal and microglial cells, astrocytes are also infected by JEV. Astrocytes are known to maintain homeostasis in the CNS and support the survival and information processing function of neurons. They respond fast to CNS infection and help regulate neuroinflammation. JEV infection results in astrocyte activation, but the infection overpowers the capacity of activated astrocytes to maintain metabolic homeostasis, resulting in an over accumulation of toxic byproducts of metabolism that are injurious to neuronal viability. However JEV infection triggers metabolic reprogramming by upregulating the expression of many proteins such as IP-10, ceruloplasmin and glutamine synthase by astrocytes, involved in the metabolic pathways vital for maintaining neuronal health. This increase is deficient to meet the increased demand that accompanies JEV infection. Astrocytes help in the transmission of JEV from peripheral tissues to the cerebrospinal fluid.

Transmission
The JE virus exists in a zoonotic transmission cycle between mosquitoes and pigs and/or water birds; humans get infected only accidentally when bitten by an infected mosquito [Fig 3] and are a dead-end host [Gould et al., 2008]. JEV has been isolated from many mosquito species in field studies, and even though the major mosquito vectors differ in diverse geographical regions, the most important is Culex tritaeniorhynchus. For Eastern Asia, Southern Asia and Southeastern Asia, the chief vector is C. tritaeniorhynchus [Rao et al., 2001]. There are two epidemiological forms of transmission: an endemic form in tropical areas with virus circulation almost throughout the year, but with a wide seasonal peak probably due to irrigation practices; and an epidemic form in more temperate areas with clear summer seasonality [Schuh et al., 2013;Gao et al., 2013]. Subsequently, JE is mainly a rural disease, where Cx. tritaeniorhynchus mosquitoes breed in rice paddies and pigs provide the main source of blood meals, with the consequence of transmission cycles in close proximity to human habitation.

Diagnosis
Patients with JE present many signs of acute encephalitic syndrome. There are various possible causes of acute encephalitic syndrome; therefore laboratory confirmation is crucial for the accurate diagnosis of JE [ Table 2], which is a tough task because of very low viremia. Reverse passive hemagglutination, immunofluorescence and staphylococcal coagglutination tests using polyclonal or monoclonal antibodies have proved the value of antigen detection in Cerebrospinal fluid (CSF) for rapid diagnosis of JE. Advanced methods like Immunogold silver staining (IGSS), have been effectually used in the detection of antigen in mononuclear cells of peripheral blood and CSF of patients. Immunohistochemistry has been used to detect viral   Table 2. Laboratory diagnostic tools for Japanese encephalitis Please use Adobe Acrobat Reader to read this book chapter for free. Just open this same document with Adobe Reader. If you do not have it, you can download it here. You can freely access the chapter at the Web Viewer here.

Treatment
Currently there is no therapy for JE [Saxena et al., 2009]. Presently, chemotherapy during JE is mainly supportive and not targeted towards JEV specifically. Interferon therapy has not proved to be a great success . Naturally occurring compounds such as arctigenin, a phenylpropanoid and rosmarinic acid, which is a phenolic compound found in many Labiatae provide protection to mice against JEV GP78 by noticeably decreasing JEV induced neuronal apoptosis, activation of microglial cells, active caspase activity and induction of proinflammatory mediators in the brains of the infected animals.
An in vivo study has shown that minocycline reduces neuronal apoptosis, activation of microglial cells, active caspase activity, proinflammatory mediators and viral titer on later stages after infection. Another compound, N-methylisatin-β-thiosemicarbazone derivative is known to inhibit JEV replication completely in vitro. Glucosidase inhibitors of the endoplasmic reticulum such as N-nonyl-deoxynojirimycin, which block the trimming step of N-linked glycosylation, have been shown to eliminate the production of many endoplasmic reticulumbudding viruses, including dengue type-II and JEV. Another recent study carried out in mice using RNA interference showed that a single intracranial organization of lentiviruses delivering short hairpin RNA or lipid-complexed small interfering RNA (siRNA) either before or after the viral challenge was sufficient to provide protection against lethal encephalitis. From the study it was clear that by precise drug design of the conserved site, a single siRNA treatment could suppress viral infection across species, thus enhancing the treatment of acute viral infections with overlapping clinical symptoms [Ghosh et al., 2009].

Protection
The control of JE is based mainly on three measures: mosquito control, avoiding human exposure and immunization. Mosquito control has failed to be an effective measure and suffers from the lack of research into new pesticides. Avoiding complete exposure from infected mosquitoes is not practically possible. Accordingly, immunization is the only effective method for long-term protection. To prevent JE, it is crucial to apply a large scale vaccination for the human population in JE prone areas . There are many groups of vaccines [ Table 3] which are presently in use such as purified, formalin-inactivated mouse brain derived and cell culture derived live attenuated vaccine [Lin et al., 2013]. Several vaccines are still in different stages of development such as DNA vaccines, recombinant virus based/chimeric vaccines . In most countries, the currently available vaccine for use is an inactivated vaccine derived from mouse brain, which is manufactured in many regional countries, but it is costly, involves three doses, needs boosting at quite frequent intervals, may be less effective due to antigenic variation and gives rise to a number of vaccine related adverse reactions [Yun et al., 2013].

Conclusion
JE is a neurological disease caused by a mosquito-borne JEV. Unlike smallpox and polio, JEV cannot be completely eradicated because of its enzootic nature of transmission. Ever since its discovery, JEV has continued to expand its activity into new regions, while many JE vaccines have been made commercially available in different parts of the world. Concern about its spread has been emphasized by the recent emergence and spread of JEV in northern Australia, making it a major concern for global public health. Currently, prevention of infection with most arboviruses relies primarily on efforts to control vector populations by spraying repellents, wearing protective clothing and reducing breeding places.
One of the most important research areas is the development of an ideal JE vaccine: one that is nontoxic, less expensive and more effective and that provides life-long protection with a single dose. The development of such a vaccine will be greatly aided by a expanding our knowledge of JEV replication and pathogenesis at the molecular level, which has now become technically possible with the use of infectious JEV SA14-14-2 cDNA technology. This technology also has huge potential for developing JEV SA14-14-2 as a vaccine vector to deliver foreign gene(s), as has already been accomplished with infectious YFV 17D cDNA technology.