Japanese Encephalitis (JE) Virus- An Overview

Japanese encephalitis (JE) virus is a mosquito-borne flavivirus that belongs to the same family as dengue, yellow fever, and West Nile viruses. It is the leading cause of vaccine-preventable encephalitis in Asia and the western Pacific, with an estimated 68,000 clinical cases and 13,600 to 20,400 deaths every year. JE virus mainly affects children, who are at higher risk of developing severe neurological complications and death.

Japanese encephalitis (JE) virus belongs to the family Flaviviridae, which includes other important human pathogens such as dengue, yellow fever, West Nile, and Zika viruses. Flaviviruses have a similar structure and morphology, which can be described as follows:

• The virus particles are spherical and about 50 nm in diameter. They consist of an electron-dense core surrounded by a lipid bilayer envelope.
• The core contains the viral genome, which is a single-stranded RNA molecule with positive polarity. The RNA is about 11 kb in length and encodes a single large polyprotein that is cleaved into 10 different proteins by viral and cellular proteases.
• The envelope contains two types of glycoproteins: the envelope (E) protein and the membrane (M) protein. The E protein is the major surface protein of the virus and mediates attachment to host cell receptors and fusion with host cell membranes. The M protein is derived from a precursor protein called prM, which assists in the maturation and assembly of the virus particles.
• The E and M proteins form 90 dimers on the surface of the virus, arranged in an icosahedral symmetry. Each dimer consists of one E protein and one M protein. The dimers are also organized into three distinct domains: domain I (DI), domain II (DII), and domain III (DIII). DI is involved in virus assembly, DII contains the fusion peptide that initiates membrane fusion, and DIII binds to host cell receptors.

The structure of JE virus is important for understanding its replication cycle, pathogenesis, and immune response. For example, the E protein is the main target of neutralizing antibodies that can prevent infection or disease. The DIII domain of the E protein is particularly immunogenic and contains several epitopes that are recognized by different types of antibodies. The M protein also plays a role in modulating the immune response by inhibiting interferon signaling and inducing apoptosis in infected cells.

The structure of JE virus can also be used for developing vaccines and antiviral drugs. For instance, some vaccines are based on recombinant E proteins or virus-like particles that mimic the structure of JE virus. Some antiviral drugs are designed to interfere with the function or interaction of the E or M proteins, such as blocking receptor binding, fusion, or maturation.

The structure of JE virus is not static but dynamic and can change under different conditions. For example, the virus can undergo conformational changes during its entry into host cells or during its maturation process. The virus can also mutate or recombine with other flaviviruses, resulting in genetic diversity and antigenic variation. These factors can affect the virulence, transmission, and immunogenicity of JE virus.

Therefore, understanding the structure of JE virus is essential for studying its biology and developing effective interventions against it.

The genome of JE virus is a monopartite, linear single-stranded RNA with positive polarity. It is about 11 kb in length and contains a single open reading frame (ORF) that encodes a large polyprotein. The ORF is flanked by 5′ and 3′ noncoding regions (NCRs) of around 100 nucleotides (nt) and 400 to 700 nt, respectively. The 5′ end of the genome has a cap structure (m7G5’ppp5’A), but lacks a polyadenylate tail at the 3′ end.

The polyprotein is translated by the host ribosomes and processed co- and posttranslationally by cellular and viral proteases into three structural proteins and seven nonstructural (NS) proteins, in the following order: C-prM-E-NS1-NS2A-NS3-NS4A-NS4B-NS5. The structural proteins form the viral particle, while the NS proteins are involved in viral replication, modulation of host immune response, and virulence.

The structural proteins are:

• C (capsid): a small (~11 kDa) basic protein that forms the nucleocapsid with the genomic RNA.
• prM (precursor membrane): a glycosylated protein (~26 kDa) that assists in the folding and maturation of the E protein and protects it from premature fusion in acidic environments. It is cleaved into M (membrane) protein during virus egress.
• E (envelope): a large (~50 kDa) glycosylated protein that forms the outer surface of the viral particle. It mediates receptor binding, membrane fusion, and hemagglutination. It also elicits neutralizing antibodies and protective immunity.

The NS proteins are:

• NS1: a glycosylated protein (~48 kDa) that is essential for viral replication. It forms a homodimer and associates with the endoplasmic reticulum membrane. It also secreted into the extracellular space, where it interacts with various host factors and modulates immune response, complement activation, and vascular permeability.
• NS2A: a small (~22 kDa) hydrophobic protein that is involved in virus assembly, budding, and antagonizing interferon signaling.
• NS2B: a hydrophilic protein (~14 kDa) that forms a stable complex with NS3 and acts as a cofactor for its protease activity.
• NS3: a multifunctional protein (~70 kDa) that has protease, helicase, NTPase, and RNA triphosphatase activities. It cleaves the polyprotein at specific sites and unwinds the viral RNA during replication.
• NS4A: a small (~16 kDa) hydrophobic protein that induces membrane alterations and serves as a scaffold for the replication complex.
• NS4B: a hydrophobic protein (~27 kDa) that also induces membrane alterations and inhibits interferon signaling.
• NS5: the largest (~100 kDa) and most conserved protein of JE virus. It has methyltransferase and RNA-dependent RNA polymerase activities. It catalyzes the capping and elongation of the viral RNA and suppresses host innate immunity.

Japanese encephalitis (JE) virus is a mosquito-borne flavivirus that is closely related to dengue, yellow fever, and West Nile viruses. JE virus is the main cause of viral encephalitis in many countries of Asia, with an estimated 68,000 clinical cases every year. The case-fatality rate among those with encephalitis can be as high as 30%, and permanent neurologic or psychiatric sequelae can occur in 30%–50% of those with encephalitis.

JE virus is transmitted to humans through the bite of infected Culex species mosquitoes, particularly Culex tritaeniorhynchus. The virus is maintained in an enzootic cycle between mosquitoes and amplifying vertebrate hosts, primarily pigs and wading birds (also referred to as natural reservoirs). Humans are incidental or dead-end hosts because they usually do not develop high enough concentrations of JE virus in their bloodstreams to infect feeding mosquitoes.

JE virus transmission occurs primarily in rural agricultural areas, often associated with rice production and flooding irrigation. In some areas of Asia, these conditions can occur near urban centers. In temperate areas of Asia, JE virus transmission is seasonal, and human disease usually peaks in the summer and fall. In the subtropics and tropics, transmission can occur year-round, often with a peak during the rainy season.

24 countries in the WHO South-East Asia and Western Pacific regions have endemic JEV transmission, exposing more than 3 billion people to risks of infection. The risk areas and transmission season for JE vary by country and may change over time due to factors such as climate change, land use, animal husbandry practices, vector control measures, and human behavior. Travelers to JE-endemic areas may be at risk of infection if they visit or stay in rural or agricultural settings where human exposure to infected mosquitoes is likely.

The replication cycle of JE virus involves several steps that take place in the cytoplasm of the host cells. The steps are as follows:

• Attachment and entry: The virus attaches to the host cell surface by binding to unknown cellular receptors and attachment factors. The virus is then internalized by clathrin-mediated endocytosis or apoptotic mimicry. The viral envelope fuses with the endosomal membrane and releases the genomic RNA into the cytoplasm.
• Translation and processing: The genomic RNA is a positive-sense single-stranded RNA that acts as a messenger RNA for the translation of a single long open reading frame (ORF) into a large polyprotein. The polyprotein is co- and post-translationally cleaved by cellular and viral proteases into three structural proteins (C, prM, and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The structural proteins are involved in the assembly of new virions, while the non-structural proteins are involved in the replication of the viral genome.
• Replication: The viral replicase complex is assembled from NS proteins, the viral RNA, and some host factors in the endoplasmic reticulum (ER) membranes. The replicase complex synthesizes a negative-sense RNA strand using the positive-sense RNA as a template. The negative-sense RNA then serves as a template for the synthesis of more positive-sense RNA strands. The positive-sense RNA can either be used for translation or packaged into new virions.
• Assembly and maturation: The C protein forms a nucleocapsid with the positive-sense RNA genome. The nucleocapsid buds into the ER lumen and acquires a lipid envelope with embedded prM and E proteins. The immature virions are transported to the Golgi apparatus, where the prM protein is cleaved by a cellular protease into M protein and a small peptide. This cleavage activates the E protein and renders the virions infectious.
• Release: The mature virions are released from the host cell by exocytosis.

The pathogenesis of JE virus involves the following steps:

• The portal of entry for the JE virus is through the bite of an infected mosquito, mainly Culex tritaeniorhynchus.
• After the bite, the virus enters the bloodstream and reaches the reticuloendothelial system (RES), where it replicates and causes a transient phase of viremia.
• During this phase, the virus may cross the blood-brain barrier (BBB) and invade the central nervous system (CNS), where it preferentially infects neurons and astrocytes.
• The virus enters the neuroparenchyma by endocytosis through vascular endothelial cells and distributes itself in various regions of the brain, such as the hypothalamus, hippocampus, substantia nigra, and medulla oblongata.
• The virus replicates in neurons and matures in the neuronal secretory system, causing direct neuronal cell death by apoptosis or necrosis.
• The virus also triggers an inflammatory response in the CNS, involving microglia activation, cytokine production, chemokine secretion, leukocyte infiltration, and complement activation.
• The inflammatory response causes further neuronal damage by inducing oxidative stress, excitotoxicity, blood-brain barrier disruption, and immune-mediated injury.

The clinical manifestations of JE vary depending on the severity of the infection and the age of the patient. Most people infected with JE virus do not develop any symptoms or only have mild symptoms such as fever and headache. However, about 1 in 250 infections results in severe neurologic illness, which usually occurs 5-15 days after infection.

The severe form of JE is characterized by acute encephalitis, which is an inflammation of the brain. The signs and symptoms of encephalitis may include:

• High fever
• Headache
• Neck stiffness
• Vomiting
• Confusion
• Disorientation
• Coma
• Seizures
• Muscle weakness
• Paralysis
• Movement disorders

Children may also experience gastrointestinal pain, diarrhea, and abdominal pain. In some cases, JE may also affect other organs such as the heart, liver, kidneys, and lungs.

The case-fatality rate of JE encephalitis can be as high as 30%, and among those who survive, 20%-50% may have permanent neurologic, cognitive, or psychiatric sequelae such as:

• Memory loss
• Intellectual impairment
• Behavioral changes
• Speech impairment
• Epilepsy
• Abnormal movements
• Central nervous system injury

The diagnosis of JE is based on clinical features, travel history, laboratory tests, and imaging studies. The treatment of JE is mainly supportive and symptomatic, as there is no specific antiviral therapy available. However, JE can be prevented by vaccination and mosquito control measures.

The laboratory diagnosis of JE virus infection is mainly based on the detection of virus-specific IgM antibodies in serum or cerebrospinal fluid (CSF) samples from patients with clinical symptoms of encephalitis. The IgM antibodies can be detected by using an IgM-capture enzyme-linked immunosorbent assay (MAC-ELISA), which is commercially available and widely used. The IgM antibodies usually appear 3 to 8 days after the onset of illness and persist for 30 to 90 days, but sometimes longer. However, the IgM antibodies may cross-react with other flaviviruses, such as dengue, West Nile, or yellow fever viruses, which can reduce the specificity of the test. Therefore, confirmatory testing with neutralization assays is recommended to verify the presence of JE virus-specific neutralizing antibodies and to discriminate between cross-reacting antibodies from closely related flaviviruses. A four-fold or greater rise in JE virus-specific neutralizing antibody titers between acute and convalescent serum specimens can also be used to confirm recent infection.

Other laboratory methods for the diagnosis of JE virus infection include the detection of viral antigen in tissue samples by immunofluorescence or immunohistochemistry, and the detection of viral RNA in serum, plasma, blood, CSF, or tissue samples by reverse transcription polymerase chain reaction (RT-PCR). However, these methods are less sensitive and less widely available than antibody testing, and they are usually performed only in fatal cases or for research purposes.

The laboratory diagnosis of JE virus infection requires specialized equipment and expertise, and it is not routinely available in most health facilities in endemic areas. Therefore, healthcare providers should contact their state or local health department or the CDC Arboviral Diseases Branch for assistance with diagnostic testing.

Unfortunately, there is no specific antiviral treatment for JE. Therapy consists of supportive care and management of complications. However, a vaccine is available to prevent disease.

Some of the supportive care measures that may relieve some symptoms are:

• Rest
• Fluids
• Over-the-counter pain medications
• Oxygen therapy
• Anticonvulsants
• Corticosteroids
• Intravenous fluids

Hospitalization for supportive care and close observation is generally required for patients with severe disease.

The prognosis of JE depends on the severity of the infection and the presence of neurological complications. Among patients who develop encephalitis, 20% – 30% die and 30%-50% of survivors continue to have neurologic, cognitive, or psychiatric symptoms.

Therefore, prevention is the best form of treatment for JE. WHO recommends that JE vaccination be integrated into national immunization schedules in all areas where JE disease is recognized as a public health issue. Other preventive measures include avoiding mosquito bites, using insect repellents, wearing protective clothing, and reducing mosquito breeding sites.

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