Japanese encephalitis (JE) is an ongoing public health concern in Asia1. As per literature, approximately 67,900 cases are reported every year2. The case fatality rate is approximately 25 per cent, and approximately 50 per cent of the cases reportedly suffer from permanent neuropsychiatric dysfunction2. Furthermore, around three billion people are estimated to be living in JE endemic areas3. JE virus (JEV), the causative agent of JE is transmitted to humans by Culex vishnui subgroup of mosquitoes. Clinical manifestations of JE include high fever, chills, headache, myalgia and confusion; approximately 75 per cent of the affected individuals typically experience seizures. Till date no specific treatment is available for JE, and vaccination is the only reliable option for its prevention and control4. In the available vaccine, most common vaccine against JEV genotype III (GIII) is used across endemic countries. The virus is widely distributed in southeast Asia. However, the increasing spread of JEV to the South Pacific region, including Australia5 indicates its dissemination potential to other parts of the world. In nature, JEV is maintained in a bird-mosquito and pig-mosquito cycle. Birds (Ardeid and Pond herons) and pigs act as the maintenance and amplifying hosts, respectively. It is anticipated that China and India could be under serious threat of JEV due to their uncontrolled population growth as well as the changing agricultural practices6.
Several countries, such as Japan, Korea and Taiwan, Thailand, Sri Lanka and Nepal, have achieved near elimination of JE incidence with a successful vaccination programme7. In addition, vaccination of pigs is also undertaken in countries such as Japan, Nepal, Taiwan and South Korea8. Although vaccination through organized piggeries has helped in reducing the disease incidence among pigs and claimed to be contributing to reducing the overall JE incidence in Japan9, its real impact remains to be proven. The prospects of vaccinating pigs in India is not an attractive option over human vaccination due to the disorganized nature of piggeries, high turnover rate of pig population and decreased efficacy of JE vaccines in piglets due to maternal antibodies3. The Government of India introduced JE vaccination in 2006 for children in the age group of one to 15 yr and later implemented it through the Universal Immunization Programme (UIP) in 201110.
However, despite this, an escalating trend of JE cases has been noticed in several States, particularly Assam, Uttar Pradesh and West Bengal. Furthermore, new cases with significant death rates [out of 103 deaths, 37 deaths were due to JE and 66 deaths were due to acute encephalitis syndrome (AES)] were recorded in Odisha in 201611. The increasing incidence of JE cases in the country underlines the need to understand the different triggering factors for this. Furthermore, there are increasing number of reports on displacement of the dominant and common JEV genotype, GIII with the GI respectively. The recent detection of JEV genotype I (GI) in Uttar Pradesh12 and West Bengal in India, and the emergence of JEV genotype V (GV) in China and Korea in 2009 warrant an urgent review of the ongoing prevention and control strategies in the country. In view of this global concern, the present communication reviews the existing knowledge on the molecular epidemiology of circulating JEV genotypes in India with a global perspective and discusses its impact on the ongoing vaccination strategy for the effective prevention and control of JE.
Genomic characteristics of Japanese encephalitis virus (JEV)
JEV is an enveloped, single-stranded and a, positive-sense RNA virus with a diameter of 40 to 50 nm in size. The virus has been classified as the JEV sero-complex under the family Flaviviridae. The viral genome is ~12,000 nucleotides in length and encodes for three structural (C, M and E) and seven non-structural (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5) proteins13. The single open reading frame (ORF) of the JEV genome is flanked by two non-coding regions (NCRs) at 5’ and 3’ ends. The 5’ end of the viral genome has a type I cap (m7GpppAmp). The mosquito-borne Flavivirus characteristically lacks the poly (A) tail at its 3’ end14. Instead, the 3’ end of the genome ends with a conserved dinucleotide CU. Among the viral encoded proteins, ‘E’ (envelope protein), an essential structural protein, is involved in virus-cell interaction and causes the generation of virus-neutralizing antibodies.
Serologically, only one serotype of JEV has been described so far. However, the analysis of ‘E’ gene region of the virus categorizes JEV into five genotypes – GI to GV15. A significant difference has been observed at the molecular level between GV and GI- GIV. The GV was characteristically found to have an insertion mutation in its 3’UTR and a 3.4 per cent difference in AA including 117 unique AA than the other known JEV genotypes16. Among the I-V genotypes of JEV identified so far, GIII had been detected as the dominant/most common genotype worldwide3. The recent trend of replacement of this dominant JEV GIII by the JEV GI raises serious concerns6 worldwide and further stresses upon the need for strengthening the surveillance system. Moreover, the significant difference observed in the nucleotide sequence of the circulating strain of JEV and the vaccine strain currently in use in Korea demonstrates the necessity of monitoring the molecular epidemiology of JEV in India for implementing effective control strategies in future. The report on the partial neutralizing potency of the GIII vaccine against the newly introduced GI genotype17 has alarmed the ongoing JE vaccination strategies in several countries.
Origin and evolution of JEV
As an RNA virus, the JEV genome is prone to mutations due to a lack of proofreading function during its replication. The overall mean evolutionary rate of JEV has been estimated to be 4.35×10−4 (3.4906×10−4–5.303×10−4) nucleotide substitutions per site per year. Among the genotypes, the mean evolutionary rate of the complete genome of JEV was found to be less for the GIII (6.029×10−4) than for GI and GII18. It has been experimentally demonstrated that the co-circulation of more than one genotype of JEV could result in recombinations19; this has been observed in the JEV isolate (K94P05) recovered from mosquitoes in Korea20. The phylogenetic analysis demonstrated the origin of JEV GV as the oldest lineage from its ancestor, believed to be evolved in the Indonesia–Malaya region, probably around 1550s18. It further diverged into GIV around 1736. It had also been hypothesized that JEV and the Murray Valley encephalitis virus (another serogroup member of the family Flaviviridae present only in Australia) has emerged from a common ancestry3. From genotype IV, GII and GIII diverged around 1821 and 1851, respectively. The GI has been predicted to have emerged from GII, approximately during the 1900s18. A recent study estimated the emergence of JEV approximately 3255 years ago, and subsequently diverged into five genotypes21. The proposed order of JEV divergence has been predicted as GV-GIV-GIII-GII-GI. The JEV GII has been named as the ‘Bennett isolate’. It was isolated from Korea in circa 1951, and found to have a close nucleotide sequence similarity with GI followed by GIII and GIV22. The JEV GIV has been found to be restricted only in Indonesia, with six isolates recovered to date23.
Global scenario
Encephalitis suggestive of JE was recorded in Japan in the 1870s, and the first isolate of JEV was recovered in 193524. Since then, several countries in South East Asia have reported JE cases with significant morbidity and mortality. Moreover, the widespread distribution of JEV has been recorded in certain non-endemic countries, e.g. Europe25,26 and Angola, Africa27. The pattern of distribution of JEV genotypes varies in different endemic areas. The JEV GI and GIII have a wide distribution in Asia, including China, Japan, Korea, India, Philippines and Vietnam. Genotype II has been found to be distributed in Malaysia, Indonesia, southern Thailand and northern Australia. The distribution of genotype IV has been found to be restricted to Indonesia and has not been detected in any of the other endemic countries3. Huang et al28 reported the co-circulation of GIII and GI of JEV between 2005 to 2008. In China, the most common occurrence of JEV GIII was documented from 1949 to 1989, subsequently, followed by the distribution of both GIII and GI29. Interestingly, a typical genotype variation was noted, reflected by the dominant occurrence of JEV genotypes, i.e. GI in 2001, 2003 and 2005, and GIII in 200430, indicating the competing behaviour of genotypes for establishment in the endemic areas. Although GII, GIII and GIV have been known to be distributed in Indonesia, a recent study conducted in Jambi reported the detection of GI in Culex gelidus mosquitoes31 indicates the ongoing strain replacement phenomena of the common JEV genotypes in this region. Apart from documenting human JEV isolates, China has recovered considerable JEV GIII isolates from the swinery32. Also, a new strain of JEV (GI) was isolated from vectors in Shanghai, which was found to be closely related to the previously detected Shandong strains in 201333. However, the study found that it was distantly related to other Shanghai strains isolated during the 2000s33. Interestingly, a genetically stable low virulent JEV (T1P1) was isolated from Armigeres subalbatus in Liu-Chiu-islet, a paddy-free area in Taiwan. It was proposed as a natural form of a live attenuated vaccine candidate34. The persistence of low virulent JEV (T1P1) in a paddy-free isolated area indicates the capacity of the virus to evolve in isolated/independent ecosystems. The Muar strain of JEV, the only representing GV strain was detected in Malaya, from a human case in 1952. It was subsequently detected in Tibet, China, in 200935 and recently in the Republic of Korea (ROK) after a long gap of 57 years from 2008 to 2011, from Culex bitaeniorhynchus mosquitoes36. The detection of GV in China and Korea indicates the widespread dispersal of the genotype and warrants strengthening of the surveillance system in the JE endemic countries. Despite the frequent reports of the strain replacement phenomenon among endemic countries, the continuous circulation of the indigenous JEV GIII and GV in Asia has been reported37. Although, in India, GIII and GI (6) have been recorded, continuous efforts are required to strengthen the surveillance mechanisms, because only limited strains have been isolated so far. Moreover, it is believed and proposed that other genotypes could also be circulating in this region, as experienced in other countries, e.g. the introduction of GIII in Australia and GII in northern China and Japan. The displacement of JEV GII in Torres Strait, Australia, which was prevalent until 1998 by GI in 200038 indicated the shifting behaviour of JEV genotypes from one place to the other. The unexpected emergence of JE even in higher altitudes of above 1000 to 3000 m a.s.l39 in Nepal and Tibet, warrants further studies to understand several factors involved in the emergence of JEV in these areas.
Genome sequence-based analyses have demonstrated the introduction of JEV only once in India as the JEV isolates analyzed were grouped into a single cluster40. From the available information, it is clear that JEV has been emerging in several countries by the frequent incursion of newer genotypes, probably introduced by migratory birds. While analyzing the data, it is evident that although JE cases have been occurring in several countries, such as Pakistan and Bangladesh3, due to a lack of appropriate molecular diagnostic facilities to determine the role of JEV in encephalitis cases, several countries have been unable to document the genomic information of JEV.
Indian status
In India, JE has remained a major public health concern, since its first recognition in Nagpur, Maharashtra, in 195241,42. Subsequently, the accumulation of serological evidence in southern India during 195543 resulted in cases being reported from nearly 16 States covering Andhra Pradesh, Assam, Bihar, Delhi, Goa, Haryana, Karnataka, Kerala, Maharashtra, Manipur, Nagaland, Punjab, Uttarakhand, Tamil Nadu, Uttar Pradesh and West Bengal44. At present, 24 States/Union Territories have been reported to be affected by JE in the country, and Uttar Pradesh alone contributed to more than 75 per cent of cases in 200745. The epidemic has resulted in severe infection all over the country, with case fatality rates approaching 50 per cent. The increasing incidence of JE in India is shown in Fig. 1.
;)
Fig. 1: Trend of JE incidence in India (2010-2017). Source: NVBDCP, India. JE, Japanese encephalitis.
After the massive outbreak reported from Tirunelveli in southern India in 1978, JEV was found to be endemic in several places in Tamil Nadu43,46-48. Apart from Tamil Nadu, JE has been documented in Andhra Pradesh and Karnataka. In contrast, it has also been found as a serious public health problem with an escalating trend of case incidence in north India49. The expanding potential of JEV is reflected in the detection of JEV isolate 04940–4 in Bhandara districts, Maharashtra, in 2002, indicating the spread of JEV into newer areas49. Based on the prM gene sequence analysis, Indian JEV isolates have been classified as GIII along with the Japanese isolates. Under the genotype III, Japanese and Indian isolates form two different clusters. Paranjpe and Banerjee50, analyzed the envelope gene of four Indian strains [P-20778 (Vellore), 733913 (Bankura), 826309 (Goa) and 691004 (Sri Lanka)] and identified four clusters. Uchil and Satchidanandam51 analyzed 107 ‘E’ gene sequences of JEVs isolated from different geographical regions of the world and categorized the Indian isolates of JEV into three groups. The first group was named ‘Vellore group’, which comprised two Vellore strains P-20778 and G8924 and the Thirunelveli isolate 782219. The ‘Bankura group’, the second group included the GP78 (Gorakhpur), 733913 Bankura isolate and the Goan isolate 826309. This group also included a Sri Lankan isolate H49778. The third group was named the ‘Nepal group’, which included the Assam isolate 7812474 and Nepal isolate B2524. The study further suggested a closer phylogenetic relationship between the Vellore group and the Nakayama strain from Japan. The Bankura group was found to be closer to the Chiang Mai strain from Thailand. Figure 2 shows the grouping of circulating JEVs in India.
Fig. 2: Distribution of JEV grouping based on partial genome Sequencing. Eruption of JEV GI in Gorakhpur, UP and West Bengal India has been shown.
Source:
https://www.freeworldmaps.net/asia/india/. JE, Japanese encephalitis, JEV, Japanese encephalitis virus
In 2005, Parida et al52 demonstrated that the JEV isolated during the Gorakhpur outbreak shared 95.6 per cent nucleotide sequence identity with the prototype JEV (Nakayama strain). Moreover, this strain had 94.6 per cent sequence identity with the first Indian JEV isolate (G8924). The study documented the circulation of the JEV G III in India. However, the isolates of JEV were found to be clustered in a separate group than other JEV isolates, isolated between 1956 and 198850. The study also explained the frequent introduction of JEV genotypes from other endemic countries through migratory birds, as was suggested12. The expanding potential of JEV (GP78) resulted in an outbreak in 2005 in north India49. Consequently, the incursion of a JEV GI closely related to the Japanese Swine JEV strain, and a strain from Korea has been detected in human cases in Gorakhpur, Uttar Pradesh12. Among the 66 JEV reverse transcriptase- (RT)-PCR-positive cerebrospinal fluid (CSF) samples collected, a significant number of genome sequences recovered from the samples (n=27) were found to be closely related to the Swine origin of JEV G1 and the remaining (n=39 samples) to JEV GIII in Gorakhpur during the outbreak. This clearly indicates the circulation of both the genotypes of JEV, i.e. GIII and GI in this region and supports the phenomenon of genotype replacement of GIII with GI in India. Similarly, in 2010, the incursion of JEV GI and the co-circulation of GIII and GI were documented in West Bengal53. The impact of co-circulation of GI and GIII in these regions and their relevance in disease epidemiology across the country need to be addressed. These instances suggest studying the potent expansion of GI in other JE endemic areas, particularly in Kerala, northeast India and Tamil Nadu.
The JEV infection in pigs, the amplifying host of JEV, may result in the abortion of stillborn piglets in herds with reproductive failure54. During 2019, the circulation of JEV GIII was found in pigs in Odisha, Assam and Manipur, the JE endemic States in the country. One isolate of JEV belonging to the GI was detected in pigs in, Odisha. The JE virus was predominantly detected in the serum (25.45, 3.74 and 1.49% in Odisha) than in tonsils (10.08, 0 and 3.7%) of pigs collected in Manipur, Assam and Odisha, respectively. Interestingly, the older pigs with ages above seven months (8.6%) were found to be relatively more infected than the younger pigs between three to seven months of age. Among the States studied, the highest prevalence of JEV was detected in Manipur (25.45% in serum and 10.08% in tonsils) than in Assam (3.75% in serum and 0% in tonsils) and Odisha (1.49% in serum and 3.7% in tonsils). The study evidently showed that although JEV GIII was found to be dominant in the circulation, the detection of GI in Odisha during 2019 again indicated the ongoing strain shifting the phenomenon of the existing GIII to GI in JE endemic areas, such as Odisha55. The change in the geographical distribution of the JEV clade has been recently visualized by isolating the JEV GIII (IVRI395A-a neurovirulent strain) from pigs with reproductive failure in Uttar Pradesh, India. As a member of the SKSS haplotype that predominantly circulates in humans and mosquitoes in India, the pig isolate was found to have 99.6 per cent identity with the Japanese strains JaOArS982 and 96 per cent with IND-WB-JE2 (JX072965) and IND-WB-JE1 (JX050179) isolated from West Bengal, India54. Further work on the phenotypic relevance of the mentioned substitutions in the swine isolate is warranted.
In addition to the pig JEV isolates, a highly virulent strain of JEV GIII (H225) has also been successfully isolated from equine source. It has a close genetic relationship with the south Indian isolate isolated from Vellore (P20778). The complete genome of H225 was successfully sequenced56. Similarly, the close homology of the most recent IND-WB-JE1 and IND-WB-JE2 isolates recovered from human cases in West Bengal in 2008 and 2010, respectively, with the P20778 isolate recovered from south India in 1958 indicated the silent evolving potential of the Vellore isolate and its possible repercussion as a rapidly emerging JEV strain in India57. Further in-depth research on the evolutionary relationships of P-20778, IND-WB-JE1 and IND-WB-JE2 and H225 strains is warranted. The zoonotic potential of the H225 strains requires further study.
The available limited data denote the changing molecular epidemiology of JEV in India and emphasize the need for systematic studies on the impact of genotype shift on the ongoing vaccination programmes in endemic countries. However, recent findings report the promising cross-protection of vaccine JEV GIII against heterologous genotypes58. Simultaneously, additional reports on the low protective efficacy of GIII (SA-14–14–2) against GV59, GI60 in humans and animals61 cannot be overlooked. The cross-protection ability of the current JEV vaccine strain, both inactivated and live attenuated against the circulating JEV genotypes has clearly shown that the low titre of neutralizing antibodies and reduced protection rendered by common GIII-based vaccines against GII and GI58,60. Despite the year-round occurrence of JE cases in Andhra Pradesh since 198162, the specific genotype involved and its characterization have not yet been studied. Furthermore, serological and entomological evidence supports the JEV activity in this region63. The pattern of distribution and diversity of JEV genotypes in Mandya and other adjoining districts in Karnataka need to be urgently addressed because this region has been identified as one of the high-risk zones for JE. The interesting paradoxical observation of frequent detection of JEV antigens in vector mosquitoes in the Tanjore area, known as ‘RICE BOWL’ of Tamil Nadu, despite the absence of clinically overt JE human cases and a low rate of JE infection in children64, warrants further systematic studies to identify and characterize the circulating JEV genotypes in this region.
Because JEV and West Nile viruses are transmitted by the same vector mosquitoes and have been found to cause human encephalitis65,66, the possible co-circulation of both the viruses and their impact on the dynamics of the circulation of JEV genotypes in India needs to be studied. Attempts to develop improved and more effective JEV vaccines are underway globally. Although the available JEV vaccines induce a similar antibody response against most strains, the low titres of the neutralizing antibody have been detected, particularly against the genotypes GI and GIV67. However, further studies are highly warranted to analyze the cross-protection effect on other genotypes such as GI and also possibly for GV, which is associated with widespread dispersal. Moreover, the impact of displacement of GIII with GI on the ongoing vaccination with respect to complete protection conferred by the GIII-based vaccine in India should be carefully evaluated because of the isolation of GI in the CSF of patients with JE in vaccinated cases documented in the Yunnan Province of China68. Considering the recent increase in the JEV activity in West Bengal and Odisha, attempts should be urgently made to understand the circulating JEV genotypes in these states. Moreover, an appropriate early warning system model may be urgently developed to contain the spread of the pathogen.
Research needs
The available data on the molecular characterization of JEV isolates provide evidence of its geographical expansion and establishes the potential of certain JEV genotypes for further spread to different countries and continents. The nucleotide sequence information of circulating JEV genotypes isolated from several endemic States should be studied in India because the available complete genomic sequences of circulating JEV in the public domain are limited in number despite the increased prevalence of JEV infection in the country since the 1970s (Table). The reason for the shift in the age group of JE cases in adults has not been known and needs further research, probably due to a lack of immunity by subclinical infection or incursion of a virus variant.
Table: Complete genome sequences of Indian Japanese encephalitis virus isolates available in the National Center for Biotechnology Information GenBank
Conclusion
The available evidence clearly indicates the rapid expansion of JEV to newer parts of the country, with several regions becoming endemic. The silent and rapid evolution of JEV could result in the potential emergence of virus variants in endemic areas as well as in newer foci, particularly Uttar Pradesh, West Bengal, Assam, Kerala, Karnataka and Tamil Nadu. The circulation of multiple JEV genotypes/strains possibly introduced by migratory birds from different parts of the world between and within the countries warrants urgent measures to study its relevance to implementing JE control strategies in India. The expanding and emerging potential of JEV strains of animal origin and their possible introduction in the country and their impact on human health in India warrants urgent studies. Strengthening the surveillance system, developing an appropriate early warning system and increasing vaccination coverage will be effective in preventing and controlling the spread of JE in India.
Acknowledgment: The authors acknowledge the Drs P. Jambulingam (former Director), M. Muniaraj, Vector Control Research Centre, Pondicherry for their valuable comments rendered. Dr A.Venkatesh is acknowledged for the illustrations, Servshree K. Venkatasubramani, S. Victor Jerald Leo, K.J. Dhananjeyan and T. Balaji are acknowledged for assisting in the collection of literature.
Financial support & sponsorship: None.
Conflicts of Interest: None.
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