Japanese encephalitis in India

1. Epidemiology of Japanese
Encephalitis in India
Dr Vinodh Kumar O.R
Division of Epidemiology
ICAR- Indian Veterinary Research Institute
Bareilly, Uttar Pradesh-243 122

2. INTRODUCTION
Japanese encephalitis virus (JEV) - arthropod-borne zoonotic viral
disease
Family: Flaviviridae
Genus: Flavivirus
Species : Japanese encephalitis virus (JEV)
Enveloped, positive sense ssRNA
Australasian countries-
About 68,000 JE cases/year
10,000-15,000 deaths/year
The virus may infect a number of other domestic animals,
including cattle, sheep, goats, dogs, and cats, in which these
infections are typically asymptomatic.

3. JE virus
The JEV genome is encoded by a plus-sense, single stranded RNA
of approximately 11 kb.
Based on E gene phylogenetic analysis, JEV strains have been
classified into five genotypes (G I-V).

4. JEV genotypes
Five genotypes (I–V) & only one serotype (Solomon et al., 2003).
JEV genotype in India
Till 2007, all isolated strain belong to GIII
GI reported:
2009: Gorakhpur region (Uttar Pradesh)
2010: West Bengal (Fulmali et al., 2011).

5. JEV genotypes
• Genotype I (GI) includes strains isolated in Southeast Asia, Australia,
northern Thailand, Cambodia, Korea, Japan, and India.
• Genotype II (GII) includes strains isolated in southern Thailand,
Malaysia, Indonesia, and Australia.
• Genotype III (GIII) includes strains isolated in Southeast Asia, Japan,
China, Korea, Taiwan, and the Central Asian sub-continent including
India.
• GIV includes strains isolated in Indonesia alone and GV includes a
single strain isolated in Singapore (Hasegawa et al., 1994; Nga et al., 2004).
• GIII is widely distributed throughout Asian countries and most of the
JEV strains isolated in India belong to GIII.
• JEV GI has recently been reported in human patients from Gorakhpur
(Uttar Pradesh, India) (Fulmali et al., 2011).

6. Global scenario
• Genetic studies, suggest that JEV originated from an
ancestral virus in the area of malay of Archipelago.
• The first outbreak of encephalitis attributed to JEV was
reported in Japan in 1871.
• Major epidemics have been reported about every ten years.
• In 1935, the prototype Nakayama strain was isolated from
the brain of a patient suffering from encephalitis.
• India, Pakistan, Nepal, Sri Lanka, Burma, Laos, Vietnam,
Malaysia, Singapore, Philippines, Indonesia, China,
maritime Siberia, Korea, and Japan - proven epidemics
(Vaughn and Hoke , 1992).
• Role of Cx. tritaeniorhynchus as vector & involvement of
ardeids & pigs as hosts demonstrated in 1938 (Tsai, 1990)
•

7. Incidence of JE in India
(Tiwari et al., 2012)
 The three southern states of Tamil
Nadu (TN), Andra Pradesh,
Karnataka were reporting higher
incidence.
 JE is emerging as a public health
problem in Kerala.
 In a few villages of Cuddalore
district of Tamil Nadu, a known
JE-endemic area (Chidambaram,
Virudhachalam, Thittakudi)
JE in Southern India

9. Indian Scenario
• In India, the fist human case was reported from North Arcot district of
Tamil Nadu in 1955.
• Until 1973, the disease was confined to southern parts of India, with low
prevalence; subsequently the disease spread to various other parts of
India.
• The fist outbreak of JE was recorded in 1973 from Burdwan and
Bankura districts of West Bengal.
• The first epidemic of JE was reported in 1978 in Uttar Pradesh.
• In India, while 24 states are endemic for JE, Uttar Pradesh contributed
more than 75% of cases during the recent past.
India- first reported -1955 from Vellore (TN)
1973 - Bengal
1978 - recorded in 21 States of India
1978 to 2009, - Gorakhpur (UP)
Genotype III - Indian subcontinent
Genotype I - Gorakhpur (UP) in 2009
West Bengal in 2010

10. Epidemiology
• Muar strain, identified from a patient in Malaya in 1951 is the
prototype strain of genotype V.
• Indian strains are similar to the Muar strain and belongs to genotype
V.
• Man is a dead-end in transmission cycle because of very low
viraemia and infection cannot be transmitted from man to
mosquitoes.
• Man to man transmission of the virus has not been reported to date
(Park, 2005).

11. Molecular epidemiology
• The phylogenetic analysis of the two JEV isolates obtained from Northeast India
placed them within genotype (G)III, where they form a subclade within the
Vellore group of Indian JEV strains and GIII JEV strain was circulating in the
northeastern region of India. This finding is important as it is contrary to the
belief that GI is gradually replacing GIII as the dominant genotype in Asia
(Khan et al., 2015).
• Isolation of JEV from stillborn piglets of 2016 and its close genetic relationship
with viruses detected at least three decades ago in humans and mosquitoes in
Japan suggests that the virus may have been circulating among Indian pigs for
several decades (Desingu et al., 2016).
• Phylogenetic analysis based on E- and C/prM genes indicated that the equine
JEV isolate belonged to genotype III and was closely related to the Vellore group
of JEV isolates from India ( Gulati et al., 2012).

12. Amplifier host
• Pig : Amplifier host of the JE virus
• Pigs are amplifying hosts because:
 High natural infection rate (98%-100 %)
 High viremia
 Viremia that remains high enough to infect
mosquitoes for up to 4 days
 Propensity for vector mosquitoes to feed on
swine
 High birth rate, providing a source of
susceptible pigs every year.
(Gresser et al., 1958)
Oro-nasal transmission in pigs
 JEV can be transmitted between pigs in the absence of
arthropod vectors
 Pigs shed virus in oro-nasal secretions & highly
susceptible to oro-nasal infection
 Tonsils-important site of replication (JEV persist for 25
days) (Ricklin et al., 2016)

13. Vectors- Mosquito
 Family - Culicidae
 Isolation of virus
 More than 25 species of genus Culex, Anopheles & Mansonia
 In India – From 19 species, 13 species of Culex & 3 species each of
Anopheles & Mansonioides
 Major vectors- Cx. tritaeniorhynchus, Cx. pseudovishnui, Cx. vishnui
 Important secondary vectors- Cx. gelidus Theobald, Cx. Whitmorei, Cx.
fuscocephala Theobald
 Possible maintenance of JE virus in nature throughout the year – Cx.
quinquefasciatus (Mariappan et al., 2014; Weaver, 2010)
(Samual, 2000)

14. JEV-Reservoirs
 Over 90 bird species known to be reservoir hosts of JEV
 Among them, wild wading birds, in particular egrets (Egretta garzetta) and
herons (Nycticorax nycticorax) of the Ardeidae family, are highly susceptible
to JEV infection
 Primary effective animal hosts: high virus titre and outstanding source of
infection for mosquitoes
 These birds forage in same rice-field sites that JE vectors undergo
development, thus allowing JEV transmission to occur.
 Duration of viraemia generally lasts between 1 and 7 days post-infection,
depending on the species (Banerjee et al., 1979; Soman et al., 1977)
 Viremia with high titres has been reported in ducks, young chickens,
pigeons and sparrows in India.
Cattle erget
Pond heron

15. Host and transmission cycle

16. Proportion of Japanese encephalitis cases contributed by Uttar
Pradesh in national figures by decade, 1978–2007
Kumari and Joshi, 2012

17. Proportion of Japanese encephalitis deaths contributed
by Uttar Pradesh in national figures by decade, 1978–
2007
Kumari and Joshi, 2012

18. Epidemiological pattern
• Prevalent in the month of July-September and October-March, abundant in
August. (Samuel, 2000; Sarkar
et al., 2012)
Two Epidemiological Pattern
1. Endemic pattern in tropics- Cases in rainy season -
July to September
 Southern areas (southern Vietnam, southern
Thailand, Indonesia, Malaysia, Philippines, Sri
Lanka, and southern India)
2. Epidemic pattern in Temperate zone- Cases in
summer season - May to October
 Northern areas (Northern Vietnam, northern
Thailand, Korea, Japan, Taiwan, China, Nepal,
and northern India)
(Tiwari et al., 2012)
In India, the state of Karnataka
experiences two epidemics each year,
with a severe form from April to July
and a milder one from September to
December along with the rest of India

19. Transmission cycle of the Japanese encephalitis virus
Besides mosquitoes, birds also spread the virus
to new geographic areas

20. Prevalence
• The annual incidence of the disease is of 30,000 to 50,000 cases.
(Ghosh and Basu, 2009)
• The disease can cause irreversible neurological damage.
• A large proportion of survivors, 30% to 60% of the cases, suffer
from long-term neurological manifestations in the form of
convulsions, tremors, paralysis, ataxia, and other such symptoms

21. Clinical signs- Humans
• IP- 5-15 days (Solomon & Vaughn, 2002)
• Varies from non specific febrile illness to severe neurological signs
• Children <10 years having greater risk of severe disease and higher CFR
(Halstead et al., 2013)
Mortality rate is approximately 25% to 30%.

22. Clinical symptoms- Humans
• JE disease manifestations can be divided into four
stages.
 Prodromal Stage : Abrupt onset of high fever
accompanied by headache, malaise, anorexia,
nausea & vomiting
 Acute encephalitic Stage : changes in level of
consciousness semi-coma or coma, convulsions,
neck stiffness & weakness of extremities. Fatal cases
progress rapidly and die
 Late stage: Defervescence, improved neurologic
sequelae in uncomplicated cases
Sequelae phase: complete recovery in mild cases,
while severe cases left with neurological deficits

23. Prodromal Stage
• Fever
• Rigors
• Headache
• Nausea
• and Vomiting
• The Prodromal stage usually lasts for 1 to 6 days.
• It can be as short as less than 24 hours or as long
as 14 days

24. Acute Encephalitic Stage
• Begins by the third to fifth day. The symptoms
include:
• Convulsions
• Altered sensorium, unconsciousness, coma
• Mask like face
• Stiff Neck
• Muscular Rigidity
• Tremors in fingers, tongue, eyelids and eyes.
• Abnormal movements of limbs
• Speech impairment

25. Later Stage
• Persistence of signs of CNS injury such as,
• Mental impairment.
• Increased deep Tendon reflexes
• Paresis either of the upper or lower motor
neuron type.
• Speech impairment
• Epilepsy, Abnormal movements, Behavior
abnormalities.

26. Virus isolation
Laboratory animals: Samples inoculated in 2-4 day old mice.
Kept under observation for 14 days; signs observed & brains of
dead or moribund mice collected, further virus identification
by cell culture
Samples: blood, CSF and brain tissue of clinical cases
 Cell culture: African green monkey kidney (Vero)
Baby hamster kidney (BHK) cells
C6/36 mosquito cell line
Samples: brain & blood taken from suspected animals, brain
suspension from mice after inoculation
(OIE, 2014)

27. Nucleic acid based assays
Reverse transcriptase Polymerase
Chain Reaction (RT-PCR)
 Real time RT-PCR
 Reverse Transcriptase - Loop
Mediated Isothermal Amplification
Assay (RT- LAMP)

28. Serological assays
For most practical purposes JE diagnosed serologically
(WHO, 2007)
 Antibodies begin to appear soon after onset of infection, but
only about 70-75 per cent of patients have IgM antibody in
specimens collected up to 4 days after onset.
 All patients have antibodies 7-10 days after onset (Han et al.,
1988).
 Ig class switching occurs with decline in IgM & rise in IgG, by
day 30 most of cases have IgG against JE virus (Burke et al.,
1985).

29. Other diagnostic tests
Virus neutralisation test (VNT)
 Haemagglutination inhibition (HI)
 Enzyme linked Immuno Sorbent Assay (ELISA)
 Latex agglutination test (LAT)
 Lateral Flow Immunoassays

30. Treatment
• There is no cure for JE and treatment is mainly supportive.
• Patients are not infectious, but should avoid further mosquito
bites.
• Antiviral agents : INF alfa-2a and diethyldithiocarbamate (a low
molecular weight dithiol). (Solomon et al., 2003)
• Mannitol might be used to reduce intracranial pressure.
• Minocycline: Reduces neuronal apoptosis, microglial activation,
active caspase activity, proinflammatory mediators, and viral titer
markedly on the ninth day after infection.
• N-methyl isatin-b thiosemicarbazone derivative : Inhibition of JEV
replication completely in vitro.

31. Prevention & control
Vector Management
Use Insect Repellent
Wear Proper Clothing to Reduce Mosquito Bites
Reduce Exposure to Mosquitoes During Peak
Biting Hours
Chemical Control:
(i) Larvicide:Temephos, an organophosphate
compound. Recommended dose for application of
Temephos (50 EC) is 1ppm (1 mg per liter of water).
(ii) Adulticide: Pyrethrum spray: It may be used in
indoor situations as space spray at a concentration of
0.1% - 0.2% @ 30-60 ml/1000 cu. ft. Malathion
fogging or Ultra Low Volume (ULV) spray.
Biological Control:
(i) Larvivorous fish
(ii) Endotoxin-producing bacteria, Bacillus
thuringiensis serotype H-14 (Bt H-14)

32. Vaccination in India
 JE vaccination campaign launched in 2006 wherein 11 most
sensitive districts in Assam, Karnataka & U.P. covered with
strategy to cover all children of 1-15 years of age in mass
vaccination drive (campaign mode) using live attenuated SA-14-
14-2 JE vaccine.
 Out of total 215 identified JE endemic districts, campaign activity
has been completed in 206 districts. JE vaccine has been
introduced as a part of Routine Immunization in these 206
districts (two doses, first at 9-12 months and second at 16-24
months)
 Adult JE vaccination: NVBDCP has identified 31 high burden
districts from Assam, U.P. & West Bengal for adult JE vaccination
(15-65 years age group)
(MoHFW, 2017)

33. JE vaccines in India
• One live attenuated vaccine SA 14-
4-2
• Two inactivated vaccines
• Vero cell culture derived SA 14-14-
2 JE vaccine (JEEV by BE India)
• Vero cell culture derived
821564XY, JE vaccine (JENVAC by
Bharat Biotech) (Kumar et al., 2012).

34. Purified, formalin-inactivated mouse-brain-derived JE vaccine
• Mouse-brain derived inactivated vaccines are based on
the
Nakayama and Beijing-1 strains (seroconversion rate
80%
to 90%).
• This is the only vaccine against JE approved by the
World Health Organization.
• It is available internationally under the Biken label.
• The Central Research Institute in Kasauli is the
manufacturer in India.
• It is available in lyophilized form, in which gelatin and
sodium glutamate are used as stabilizers, and thimerosal
is used as a preservative.

35. Vaccine based on the SA14-14-2 strain
• This is an attenuated and genetically stable strain that in
large-scale case–control studies in China has shown 95% protection
after two doses with an interval of one year (Diagana et al., 2007)

36. Inactivated hamster kidney cell-culture-derived JE
vaccine
• This vaccine is based on the Beijing-3 strain of JEV.
• It has relatively fewer side effects and is easy to
manufacture.
• In the last decade, a Vero cell-culture based inactivated
vaccine using various local JEV isolates has also been
developed and is undergoing clinical trials.
• A Vero-cell culture derived formalin inactivated vaccine is
being developed using an attenuated SA14-14-2 strain,
and it has induced high titers of neutralizing antibodies in
mice after two injections.
• Recently, Vero-cell culture derived formaldehyde
inactivated JE vaccine using P20778 (Indian isolate) has
been developed.

37. JE in animals
• In a study conducted in Bareilly region of U.P., the highest HI positivity
against JE was found in dogs (55.77%) followed by pigs (40%), horses
(37.65%), buffaloes (21.92%), goats (17.86%), sheep (2.38%) and cattle
(1.98%) (Mall et al., 1995).
• In Chandigarh, the seroprevalence of JE in pig was 30.3 per cent by
haemagglutination inhibition (HI) antibodies and 12.5 per cent by
complement fixation (CF) antibodies (Ratho et al., 1999).
• In Tamil Nadu, seroepidemiological studies revealed highest incidence of JE
in pigs (26.4%), followed by birds (9.37%), cattle (6.86%) and buffaloes
(5.06%) (Kumanan et al., 2002).
• Seroprevalence of JE in pigs in Haryana was found to be 18 per cent
(Nagaleelavathi et al., 2008).
• Out of 3,286 equine serum samples tested, 327 (10%) were positive for JE
antibodies according to both HI and VNT results (Gulati et al. , 2011).

38. JE in horses
• Antibodies against JEV were detected in 67 out of 637 (10.5%) horses
screened between 2006 and 2010 ( Gulati et al., 2012).
Japanese encephalitis seroprevalence in equines from 11
districts of the Haryana state (India) between 2006 and 2010