**4. 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 func‐ tioning 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 inflam‐ mation, 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 neuro‐ inflammation. 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 transmis‐ sion of JEV from peripheral tissues to the cerebrospinal fluid.


**Table 1.** Duration, signs and symptoms of Japanese encephalitis

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 [Li *et al*., 2012; Zhang *et al*., 2012]. The functions of NS3 and NS4 are prominent, they code for serine protease and RNA dependent RNA polymerase (RdRp) [Lu *et al*., 2013]. 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

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 func‐ tioning of the BBB, peripheral inflammatory cells are recruited to the infected brain that extends

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

[Anantpadma *et al*., 2011; Mastrangelo *et al*., 2012].

**4. Pathogenesis**

6 Trends in Infectious Diseases

the neuronal impairment.

JE usually develops in patients after an incubation period of 5-15 days [Table 1]. In humans, most JEV infections are asymptomatic, with about 1 in 300 JEV infections resulting in symp‐ toms ranging from non-specific mild fever to severe meningoencephalitis categorized by fever, lessened consciousness, seizures and focal neurological signs. At later stages, poliomyelitislike flaccid paralysis and parkinsonian syndrome develop, which exhibit the standard description of JE like dull, flat and mask-like face with wide, unblinking eyes, tremor, wide‐ spread hypertonia, cogwheel rigidity and other irregularities in movement [Dutta *et al*., 2010]. Paralysis of the upper body is more common than that of legs. Nearly 30% of survivors have genuine persistent motor deficits and approximately 20% have severe cognitive and language impairment [Mackenzie *et al*., 2004].
