**12. Diagnosis**

JEV infection and also their depletion affects the humoral and cellular, immune defence

Inhibitory effect of RNAi on JEV replication has been thoroughly studied *in vitro* and *in vivo* (Murakami ET AL., 2005). It is also reported that defective interfering (DI) RNA aids in the persistence of JEV (Yoon et al., 2006). Effectiveness of using siRNA expression based vectors targeting the JEV NS5 gene to inhibit JEV replication, viral protein expression, and RNA lev‐ els of JEV E-protein is hot topic of research nowadays. Several studies demonstrate that shRNAs targeting the NS5 gene could specifically and efficiently inhibit JEV replication. Many researchers have shown that siRNA/shRNAs targeting the RdRP coding gene could efficiently inhibit viral replication; the inhibition of viral replication triggered by siRNA/ shRNA targeting of the RdRP gene are reported to be more efficient compared to other genes from the genome (Neyts et al., 1999). Therefore, the NS5 gene which is highly con‐ served among different strains is often employed as an RNAi target for different studies. shRNAs targeting NS5 gene in the JEV genome are shown to be capable of interfering with JEV replication with very high specificity and efficiency. Hence shRNAs could be used as a potential tool against JEV replication *in vitro*. More research investigating RNAi methodolo‐ gies to prevent infection or reduce viremia is necessary which may lead to the development of antiviral compounds that are efficacious and inexpensive against Japanese encephalitis

IFN-α and IFN-β, play important role in recovery from flaviviral infections. However, they fail sometimes due to ability of JEV to inhibit the JAK-STAT (Janus kinase signal transducer and activator of transcription) pathway (Lin et al., 2006). Studies of DEN-2 antagonism of STAT1 phosphorylation have revealed NS4B as the primary and impor‐ tant antagonist. Still the exact mechanism of IFN antagonism is under study. The specific receptor complex for each IFN-α and IFN-β is composed of two major subunits and sev‐ eral JAK tyrosine kinases constitutively associated with the receptor. Jak1 and Jak2 are required for IFN-α/β signaling. Following binding of the receptor subunits by IFN, the JAKs trans-phosphorylate each other and then phosphorylate critical tyrosine residues within the intracellular domains of the receptor subunits (Lin et al., 2004). These phos‐ phorylated residues serve as recruitment sites for STAT proteins, which bind the activat‐ ed receptor and are in turn phosphorylated by the JAKs. The phosphorylated STAT proteins then form homodimers, or heterodimers, with other STAT proteins and translo‐ cate to the nucleus, where they bind specific DNA sequences within the promoter re‐ gions of IFN-stimulated genes (ISGs) (Fig. 5). ISG expression induces an antiviral state within the cell, can modulate cell proliferation and cell death, and modulates immune re‐

against flavivirus infections (Müllbacher et al., 2003).

**10. RNAi effect on JE**

170 Encephalitis

infections (Qi et al., 2008).

**11. JAK-STAT pathway for JE**

Serology is an important tool for the diagnosis of JE since the virus is difficult to isolate from clinical samples. The hemagglutination inhibition assay also is used but it has prac‐ tical limitations as it requires paired serum samples from the acute and convalescent phases. The IgM antibody capture ELISA for CSF and serum samples is currently the standard test for diagnosis of JE but still has the drawback of not being able to diagnose about the infection in early stage. Molecular methods using reverse-transcriptase (RT) - PCR techniques have proved to be highly effective for diagnosing infection by RNA vi‐ ruses. JE viral genome sequences have been detected by RT-PCR in CSF from acute encephalitis cases from several places around the globes. The conventional RT-PCR has shown good specificity in the diagnosis of JEV in both blood and CSF samples but it has poor sensitivity as the virus is often cleared from the peripheral circulation/CSF by the time the test is performed. With the advent of monoclonal antibodies as potential diag‐ nostic tool (Chávez *et al.,* 2010), the rapid detection of JE antigen in cerebrospinal fluid has become possible. The different diagnostic tests have been given in Table 2. However, the most rapid and potential diagnostic tool for JE diagnosis have been shown to be MAC-ELISA (Robinson *et al.,* 2010) and indirect fluorescent antibody. MRI of the brain can also be used in diagnosis. MRI changes can be co-related (Misra *et al.,* 2011) with the type of encephalitis and duration of illness.

VAX WN-immunized mice produced IFN-α and displayed increased IFN-stimulated gene transcription (Winkelmann et al., 2012). Multiple vaccines exist to control Japanese encepha‐ litis (JE), but all suffer from problems but this new flavivirus vaccine, a pseudoinfectious vi‐ rus (RepliVAX WN) that expresses the JE virus (JEV) prM and E proteins prevents flaviviral disease. Engineered second-generation RepliVAX (RepliVAX JE.2) elicited neutralizing anti‐ bodies in experimental mice and provided 100% protection from a lethal challenge with JEV

Japanese Encephalitis Virus: The Complex Biology of an Emerging Pathogen

http://dx.doi.org/10.5772/54111

173

Although a licensed vaccine has been available to prevent JE for over 40 years, approxi‐ mately 20,000 cases are reported annually with 6000 resulting in death. Unfortunately, due to gaps in surveillance, the incidence of JE is also likely to be much higher than re‐ ported. A number of different vaccines are available to prevent JE and these have dem‐ onstrated an excellent record of efficacy throughout their history. The vaccine that has been in use the longest is the INV prepared from JEV infected mouse brains. This vac‐ cine has been used extensively in East Asia since the 1960s to control JE, and is widely used throughout the world to immunize travellers who visit endemic areas, its protective efficacy is reported to be 80–90% in JEV endemic regions. But still it has a drawback that the product requires a three dose vaccination schedule in order to induce protective im‐ munity and this, along with the recommendation of boosters every 2–3 years which poses as quite expensive for nominal patients from low income group countries and time-consuming too. Furthermore they are also reported for causing allergic reactions and more dangerous side effects like complications including severe neurological disor‐ ders such as acute disseminated encephalomyelitis, etc in people being administered by

Through vaccination in the last five year, JE has been effectively controlled and eliminated in China, Japan, Taiwan, and Korea (Chung et al., 2007; Takahashi et al., 2000; Jelinek et al., 2009). Second generation recombinant vaccines are also being developed, where genes en‐ coding prM and E proteins are packed into vectors. DNA based JEV vaccines which may be very efficient against the virus are under clinical trials. DNAzymes cleave the RNA se‐ quence of the 3'-NCR of JEV genome *in vitro*, on intra-cerebral administration in JE infected mice almost completely (Appaiahgari et al., 2007) and inhibit virus replication in the brain.

There is no specific treatment or anti-viral agent for JEV infection, it is proving to be a per‐ sistent threat. Monoclonal antibodies (Yamanaka et al., 2010), corticosteroids, interferonα-2a or ribavirin were not that effective in clinical outcome. The effect of rosamarinic acid (RA) has been shown as an effective anti-viral agent that reduces JE viral load along with proin‐ flammatory cytokines in experimental animal. Neutrophils have been also shown to have degradative effect on JEV. Usage of anti-sense molecules (vivo-morpholino) directed against the viral genome, in combating the virus through inhibiting viral replication has been dem‐

Use of neutralizing bodies for vaccine designing may also serve the process.

(Ishikawa et al., 2008).

the vaccine (Widman et al., 2008).

**14. Treatment, prevention and control**

### **13. Vaccines: Immunization against JE**

Immunization against JE is cost effective strategy for control and prevention of JE. It has been reported globally that there is a decrease in incidence rates of JE in endemic areas which are administered with high immunization. The 3 most important types of JE vac‐ cines, administered in current era are: the mouse brain derived, purified, inactivated vac‐ cine based on either the Nakayama or Beijing strains of the JE virus; the cell culture derived inactivated JE vaccine based on the viral Beijing P3 strain and the cell culture derived live attenuated JE vaccine based on the SA 14‐14‐2 strain of the JE virus. In JEV infection, the immunity against prM, E and NS1 proteins is more effective than that of other viral proteins in host defense. (Gao et al., 2010). Currently available vaccines against JE include chemically inactivated vaccines (INV) and a live attenuated vaccine (LAV). Although a mouse brain derived INV produced by BIKEN had been the only in‐ ternationally approved vaccine and has been used worldwide since the 1960s. But it had a drawback as there were reports of severe adverse events including acute disseminated encephalomyelitis (ADEM) in people vaccinated with it. In early 2009, Vero cell derived INVs produced by Intercell (Austria) and BIKEN (Japan) were licensed. Although these INVs are useful in developed markets, INVs are not ideally suited for nationwide vacci‐ nation programs for many endemic countries, since INVs require multiple doses to in‐ duce long lasting immunity. LAVs are thus a useful alternative and have been used for decades in China, and other Asian countries, but their substrate and the production methods have still not been approved in other markets, which serve as a drawback to this vaccine. (Ishikawa et al., 2011). Live-attenuated virus vaccines (LAVs) and inactivat‐ ed virus vaccines (INVs) serve against flaviviral disease, they are potent and economical but do not suit immunocompromised patients. INVs are safer, but are more expensive to produce and less potent. Hence there is an immense need of devicing new and im‐ proved products.

Type I IFNs are critical for controlling pathogenic virus infections and can enhance immune responses. Hence their impact on the effectiveness of live-attenuated vaccines involves a bal‐ ance between limiting viral antigen expression and enhancing the development of adaptive immune responses. The influence of type I IFNs on these parameters has been examined fol‐ lowing immunization with RepliVAX WN, a single-cycle flavivirus vaccine (SCFV). Repli‐ VAX WN-immunized mice produced IFN-α and displayed increased IFN-stimulated gene transcription (Winkelmann et al., 2012). Multiple vaccines exist to control Japanese encepha‐ litis (JE), but all suffer from problems but this new flavivirus vaccine, a pseudoinfectious vi‐ rus (RepliVAX WN) that expresses the JE virus (JEV) prM and E proteins prevents flaviviral disease. Engineered second-generation RepliVAX (RepliVAX JE.2) elicited neutralizing anti‐ bodies in experimental mice and provided 100% protection from a lethal challenge with JEV (Ishikawa et al., 2008).

Although a licensed vaccine has been available to prevent JE for over 40 years, approxi‐ mately 20,000 cases are reported annually with 6000 resulting in death. Unfortunately, due to gaps in surveillance, the incidence of JE is also likely to be much higher than re‐ ported. A number of different vaccines are available to prevent JE and these have dem‐ onstrated an excellent record of efficacy throughout their history. The vaccine that has been in use the longest is the INV prepared from JEV infected mouse brains. This vac‐ cine has been used extensively in East Asia since the 1960s to control JE, and is widely used throughout the world to immunize travellers who visit endemic areas, its protective efficacy is reported to be 80–90% in JEV endemic regions. But still it has a drawback that the product requires a three dose vaccination schedule in order to induce protective im‐ munity and this, along with the recommendation of boosters every 2–3 years which poses as quite expensive for nominal patients from low income group countries and time-consuming too. Furthermore they are also reported for causing allergic reactions and more dangerous side effects like complications including severe neurological disor‐ ders such as acute disseminated encephalomyelitis, etc in people being administered by the vaccine (Widman et al., 2008).

Through vaccination in the last five year, JE has been effectively controlled and eliminated in China, Japan, Taiwan, and Korea (Chung et al., 2007; Takahashi et al., 2000; Jelinek et al., 2009). Second generation recombinant vaccines are also being developed, where genes en‐ coding prM and E proteins are packed into vectors. DNA based JEV vaccines which may be very efficient against the virus are under clinical trials. DNAzymes cleave the RNA se‐ quence of the 3'-NCR of JEV genome *in vitro*, on intra-cerebral administration in JE infected mice almost completely (Appaiahgari et al., 2007) and inhibit virus replication in the brain. Use of neutralizing bodies for vaccine designing may also serve the process.
