VECTOR-BORNE DISEASES, SURVEILLANCE, PREVENTION Longitudinal Studies of Japanese Encephalitis Virus Infection in Vector Mosquitoes in Kurnool District, Andhra Pradesh, South India N. ARUNACHALAM,1 U.S.N. MURTY,2 D. NARAHARI,3 A. BALASUBRAMANIAN, P. PHILIP SAMUEL, V. THENMOZHI, R. PARAMASIVAN, R. RAJENDRAN, AND B. K. TYAGI Centre for Research in Medical Entomology (Indian Council of Medical Research), 4, Sarojini Street, Chinna Chokkikulam, Madurai, Tamil Nadu, India KEY WORDS Japanese encephalitis, Culex tritaeniorhynchus, Culex gelidus, Kurnool, south India Japanese encephalitis virus (family Flaviviridae, genus Flavivirus, JEV) is currently one of the most important arboviral childhood viral encephalitis in Asia, causing at least 50,000 clinical cases and 10,000 deaths every year (WHO 2005). Japanese encephalitis (JE) is a zoonotic disease, with a complex life cycle involving pigs and ardeid birds and vector mosquitoes. Humans are only occasionally infected and are a “dead end” host, because viremia in human blood is too low and transient to infect mosquitoes. JE is predominantly a rural disease, and it occurs scattered over extensive areas and seldom in peripheral localities of cities (Reuben and Gajanana 1997). In India, JE was Þrst reported in 1955 (Work and Shah 1956); subsequently, many epidemics have occurred in different parts of the country. JE outbreaks have been reported as many as 25 states/union territories of India (Kabilan et al. 2004). In some states, such as Uttar Pradesh, Bihar, and Andhra Pradesh, JE has emerged as a perennial public health problem during the rainy season. In 2005, a major encephalitis outbreak was reported in Gorakhpur, Uttar Pradesh, which was the most severe JE epidemic so far reported, affecting ⬎5,700 persons, mainly children, with ⬎1,300 deaths (WHO 2006). JE virus has been recovered from several mosquito species (19 species) in different parts of India, and the most important vectors are Culex tritaeniorhynchus Giles and Culex vishnui Theobald from which the largest number of isolations have been made (Geevarghese et al. 2004). In Asia, development of irrigation systems and the expansion of rice (Oryza sativa L.)-growing areas have facilitated the increase of JEV vectors (Lacey and Lacey 1990). Although epidemics invariably are preceded by increase in vector abundance, several other factors, including mosquito infection rate are involved (Gajanana et al. 1997). Our knowledge of the epidemiology of JE virus is still incomplete considering the diverse ecogeographical regions in India. A detailed understanding of JE epidemiology will lead to the achievement of prediction of outbreaks and sound mosquito control methods. The present studies were undertaken to investigate the dynamics JEV transmission between June 2002 and July 2006 in a JE-endemic district, Kurnool, Andhra Pradesh (AP) state, south India. The main objective was to determine the seasonal abundance and JE infection rates of mosquito vector population to understand their role in the transmission of JE. Materials and Methods Corresponding author, e-mail: crmeicmr@icmr.org.in. 2 Indian Institute of Chemical Technology, Hyderabad, Andhra Pradesh, India 500007. 3 Department of Health, Andhra Pradesh, India 500001. 1 Study Area. The study was carried out in Kurnool district, AP (Fig. 1), and its total population is 1,724,795. Most of the people are working in the ag- 0022-2585/09/0633Ð0639$04.00/0 䉷 2009 Entomological Society of America Downloaded from https://academic.oup.com/jme/article/46/3/633/861274 by guest on 13 February 2022 J. Med. Entomol. 46(3): 633Ð639 (2009) ABSTRACT A 4-yr (2002Ð2006) entomological study was carried out in Kurnool district, Andhra Pradesh state, south India, to identify the mosquito vectors of Japanese encephalitis virus (family Flaviviridae, genus Flavivirus, JEV). In total, 37,139 female mosquitoes belonging Þve genera and 18 species resting on vegetation were collected in villages and periurban areas at dusk. Mosquito species composition and pattern of JEV infection in mosquitoes varied in periurban and rural areas. In periurban area, Culex gelidus Theobald was abundant, msking up 49.7% of total catch followed by Culex tritaeniorhynchus Giles (44.5%). In rural area, Cx. tritaeniorhynchus was predominant, making up 78.9% of total catch followed by Culex quinquefasciatus Say (10.8%), Anopheles subpictus Grassi (7.1%), and Cx. gelidus (1.1%). In light trap collections, Cx. gelidus and Cx. tritaeniorhynchus predominated in periurban and rural areas, respectively. Of 50,145 mosquitoes screened JEV isolations were made only from Cx. gelidus and Cx. tritaeniorhynchus. Based on high abundance and frequent JEV isolation, Cx. tritaeniorhynchus was found to be the principal vector in both areas, whereas Cx. gelidus plays a secondary vector role in periurban areas only. 634 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 46, no. 3 ricultural sector, are of low-income, and live in substandard housing. Domestic animals such as cattle, pigs, and poultry are commonly sharing the living space with the human population. A single crop of rice was raised in irrigated Þelds of study villages from August to January. The climate of Kurnool is topical, and it can be divided into three seasons: dry (FebruaryÐJune), wet or monsoon (JulyÐOctober), and winter (NovemberÐJanuary). Southwest monsoon gives more dependable rains in the area, which begins in June and persists through October. During the study period (2002Ð2006), the maximum temperature ranged from 30.3⬚C (December 2005) to 42.8⬚C (May 2003) (Fig. 2). Two suburban localities and six villages where at least one JE case occurred during 1997Ð2002 were selected as index areas and sampled once every 2 mo. The suburban localities located on the margin of Kurnool are deÞned as periurban areas, which are outside the municipal boundaries and thus without basic civic amenities. Mosquitoes resting on vegetation and bushes around cattle sheds were collected by three insect collectors for 1 h after dusk by mouth aspirator. Two cattle sheds were sampled in each area, and the same sheds were sampled on each occasion by three insect collectors. Mosquito (only females) abundance was calculated as number collected per person per hour. Adult mosquitoes were also collected by using light traps outdoors for 1 h simultaneously, and the samples were also used for virus isolation. Mosquitoes were transported to the laboratory for identiÞcation and enumeration. Downloaded from https://academic.oup.com/jme/article/46/3/633/861274 by guest on 13 February 2022 Fig. 1. (A) Map of India showing location of the Kurnool district, AP, India. (B) Map of Kurrnool district showing location of study areas. May 2009 ARUNACHALAM ET AL.: JE VIRUS INFECTION IN VECTOR MOSQUITOES 635 350 80 60 250 Rainfall (mm) 50 200 40 150 100 20 50 10 0 JUN JULY AUG SEP OCT NOV DEC JAN FEB MAR APR MAY JUN JULY AUG SEP OCT NOV DEC JAN FEB MAR APR MAY JUN JULY AUG SEP OCT NOV DEC JAN FEB MAR APR MAY JUNE JULY AUG SEPT OCT NOV DEC JAN FEB MAR APR MAY JUNE JULY 0 2002 2003 TOTAL Rainfall (mm) 2004 Max. Temp. 2006 2005 Min. Temp. Relative Humidity 17.30hrs Fig. 2. Meteorological data recording during the study. Female mosquitoes were tested in single-species pools of 3Ð50 for JE virus by using an antigen-capture enzymelinked immunosorbent assay (ELISA) for the initial screening for ßavivirus and inoculation of Toxorhynchites splendens Wiedemann combined with an indirect immunoßuorescence assay (Toxo-IFA) to conÞrm infection with JEV (Gajanana et al. 1997): 1) antigen-capture ELISA by using monoclonal antibody 6B4A-10 (reactive against all the viruses in the JE-WN-SLE-MVE complex) as capture antibody and monoclonal antibody peroxiTable 1. dase conjugate SLE MAB 6B6C-1 (reactive against all ßaviviruses) as detector antibody, and 2) insect bioassay by using mosquito pools positive in ELISA inoculated intracerebrally into Tx. splendens larvae (early stage of third instar). In total, eight larvae from a laboratory cyclic colony were used for each positive pool, and ⬇0.17 ␮l of ELISA-positive mosquito pools was inoculated. Inoculated larvae were incubated at 29⬚C for 7 d, and head squashes of inoculated larvae were tested by IFA by using a JEV-speciÞc monoclonal antibody, MAB 112 Species composition of mosquitoes in Kurnool Species Aedes aegypti (L.) Ae. albopictus (Skuse) Ae. vexans (Meigen) Ae. vittatus Anopheles barbirostris An. hyrcanus An. peditaeniatus An. stephensi Liston An. subpictus Grassi An. vagus Armigeres subalbatus C.x gelidus Theobald Cx. infula Theobald Cx. pseudovishnui Cx. quinquefasciatus Say Cx. tritaeniorhynchus Giles Cx. vishnui Theobald Lutzia fuscanus Wiedemann Mansonia indiana Edwards Ma. uniformis Theobald Total Resting collection at dusk Light trap Rural Periurban Rural Periurban Total collected 31 0 0 0 77 42 18 26 1,531 4 99 237 0 0 2315 16,948 124 17 0 1 21,470 29 3 4 0 3 6 5 79 295 1 48 7,793 0 9 363 6,979 25 6 1 20 15,669 3 0 0 1 98 54 18 0 1,134 1 78 194 1 0 1,673 4,405 0 0 0 0 7,660 16 0 1 1 6 8 0 14 405 0 66 11,602 0 3 491 4,388 20 0 1 27 17,049 79 3 5 2 184 110 41 119 3,365 6 291 19,826 1 12 4,842 32,720 169 23 2 48 61,848 % 0.13 0.005 0.008 0.002 0.3 0.18 0.07 0.19 5.44 0.01 0.47 32.06 0.002 0.02 7.83 52.9 0.27 0.04 0.002 0.08 Downloaded from https://academic.oup.com/jme/article/46/3/633/861274 by guest on 13 February 2022 30 Temperature 0C / Relative Humidity (17.30hrs) 70 300 636 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 46, no. 3 500 4 3.5 3 350 2.5 300 2 250 200 1.5 150 1 Minimum infection rate / 1000 400 100 0.5 50 0 Ju l. Se p. N o Ja v. n. 05 M ar . M ay . Ju l. Au g. Se p. N ov Ja . n. 06 M ar . M ay . Ju l. ar . ay . M M Ju n e 02 Se p. D ec Fe . b. 03 Ap r. Ju l. Au g. Se p. O ct . N ov . D e Ja c. n. 04 0 Month Urban MIR Fig. 3. Urban PMH Vector abundance and JEV infection of Cx. tritaeniorhynchus in rural areas of Kurnool district. (Kimura-Kuroda and Yasuri 1983), and virus was detected by anti-mouse immunoglobulin conjugated with ßuorescein isothiocyanate (Dakoppats, Glostrup, Denmark). Virus infection rate in mosquitoes was expressed as minimum infection rate (MIR) per 1,000 females tested (Chiang and Reeves 1962). The pool size of adult females for virus isolations varied from 3 to 50 specimens per pool. MIR is not an appropriate parameter for expressing infection rates and for comparison between vector species when pool sizes are unequal. To overcome this problem maximum likelihood estimate method was also used to calculate the infection rate (http://www. cdc.gov/ncidod/dvbid/westnile/software.htm). Virus infection rate was expressed as maximum likelihood estimate with 95% conÞdence intervals. Results Mosquito abundance varies in periurban and rural ecosystems. In total, 37,139 female mosquitoes representing six anopheline and 12 culicine species were collected in dusk collections (Table 1). In periurban areas, Cx. gelidus was the most abundant species, making up 49.7% of the total mosquitoes collected, followed by Cx. tritaeniorhynchus (44.5%), whereas in rural areas Cx. tritaeniorhynchus was the most abundant species, making up 78.9% of the total collected, and Cx. gelidus consisted only 1.1% of the total catch. In light trap collection, 24,709 female mosquitoes representing six anopheline and 12 culicine species were collected. Cx. gelidus was the most abundant species in periurban areas, making up 68.1% of the total mosquitoes collected, followed by Cx. tritaeniorhynchus (25.7%), whereas in rural areas Cx. tritaeniorhynchus was the most abundant species, making up 57.5% of the total mosquitoes collected, and Cx. gelidus consisted only 2.5% of the total. Cx. tritaeniorhynchus population ßuctuates in a similar pattern in both rural and periurban areas (Figs. 3 and 4). Abundance of Cx. tritaeniorhynchus was lowest in summer, and it increased from September onward coinciding with monsoon and rice cultivation. Cx. tritaeniorhynchus abundance did not show signiÞcant correlation with meteorological factors except for temperature in rural and periurban areas. Correlation analysis of abundance of Cx. tritaeniorhynchus with temperature showed a signiÞcant negative relationship in rural areas (r ⫽ ⫺0.50, P ⬍ 0.01) and in periurban areas (r ⫽ ⫺0.49, P ⬍ 0.01). Abundance of Cx. gelidus did not show any major seasonal ßuctuations in periurban areas (Fig. 5). Altogether, 951 pools (30,264 specimens) of Cx. tritaeniorhynchus, 594 pools (19,245 specimens) of Cx. gelidus, seven pools (142 specimens) of Cx. vishnui, two pools (11 specimens) of Cx. pseudovishnui, 14 pools (483 specimens) of Anopheles subpictus were tested for ßavivirus infection. JEV was isolated only from Cx. tritaeniorhynchus and Cx. gelidus. Cx. tritaeniorhynchus yielded more isolates (19), with an overall MIR of 0.63, followed by Cx. gelidus (11) with an MIR of 0.57. Minimum infection rate of Cx. tritaeniorhynchus ranged from 1.03 to 3.18 in rural areas (Fig. 3) and from 0.72 to 3.70 in periurban areas (Fig. 4). Infection in Cx. gelidus was observed only from periurban areas, and the MIR ranged from 0.17 to 23.26 (Fig. 5). Maximum likelihood estimates of JEV infections for Cx. tritaeniorhynchus and Cx. gelidus were calculated. Maximum likelihood estimates of JEV infections of Cx. tritaeniorhynchus were almost similar in periurban 0.64 and rural areas (0.63). Cx. tritaeniorhynchus showed a higher rate of infection (0.64) Downloaded from https://academic.oup.com/jme/article/46/3/633/861274 by guest on 13 February 2022 PMH (female Cx. tritaeniorhynchus collected per man-hour) 450 637 140 3.5 120 3 100 2.5 80 2 60 1.5 40 1 20 0.5 Minimum infection rate / 1000 ARUNACHALAM ET AL.: JE VIRUS INFECTION IN VECTOR MOSQUITOES 0 Ju l. Se p. N o Ja v. n. 05 M ar . M ay . Ju l. Au g. Se p. N ov Ja . n. 06 M ar . M ay . Ju l. ar . ay . M M Ju n e 02 Se p. D ec Fe . b. 03 Ap r. Ju l. Au g. Se p. O ct . N ov . D e Ja c. n. 04 0 Month MIR Fig. 4. PMH Vector abundance and JEV infection of Cx. tritaeniorhynchus in periurban areas of Kurnool district. than Cx. gelidus (0.56). However, 95% conÞdence intervals of the two species overlap; hence, the difference is not signiÞcant (Table 2). Discussion The seasonal distribution of vector mosquitoes varies in time and space depending upon environmental conditions and availability of breeding habitats. The study sites (rural/periurban) with different ecological conditions show striking differences in relative abun- dance of JE vectors. Cx. tritaeniorhynchus populations were more abundant during the monsoon and immediate postmonsoon seasons due to the availability of paddy Þelds (only rural) and rainwater pools. Because these breeding habitats were absent during summer, Cx. tritaeniorhynchus showed a negative relationship with temperature. It shows Cx. tritaeniorhynchus population is strongly affected by the availability of suitable breeding habitats. Cx. gelidus was the predominant species in periurban areas, and no seasonal pattern on population density was observed, unlike for 25 500.0 350.0 15 300.0 250.0 200.0 10 150.0 5 100.0 50.0 e Se p. D ec Fe . b. 03 Ap r. Ju l. Au g. Se p. O ct . N ov . D ec Ja . n. 04 M ar . M ay . Ju l. Se p. N ov Ja . n. 05 M ar . M ay . Ju l. Au g. Se p. N ov Ja . n. 06 M ar . M ay . Ju l. 0 02 0.0 Month MIR Fig. 5. PMH Vector abundance and JEV infection of Cx. gelidus in periurban areas of Kurnool district. Minimum infection rate /1000 20 400.0 Ju n PMH (female Cx. gelidus collected per manhour) 450.0 Downloaded from https://academic.oup.com/jme/article/46/3/633/861274 by guest on 13 February 2022 PMH (female Cx. tritaeniorhynchus collected per man-hour) May 2009 638 JOURNAL OF MEDICAL ENTOMOLOGY Table 2. Vol. 46, no. 3 Maximum likelihood estimate of JE virus infection in Cx. tritaeniorhynchus and Cx. gelidus Mosquito pools positive by IFA/examined (no. female specimens) Parameter JE virus infection (IFA) Maximum likelihood estimate 95% conÞdence intervals Cx. tritaeniorhynchus Cx. gelidus Rural Periurban Rural Periurban 12/617 (19,179) 0.63 0.34, 1.07 7/334 (11,085) 0.64 0.28, 1.26 0/20 (389) 0 11/574 (18,856) 0.56 0.29, 0.97 the 2003 animal census, which is quite high compared with other states of India. Pigs were maintained in small groups in backyard pens. Consequently, JE vectors and the amplifying hosts were in proximity, favoring the transmission of JEV to humans. Acknowledgments We thank the Director General, Indian Council of Medical Research, New Delhi, for encouragement and facilities. The technical assistance rendered by Shriyuts C. Sundararaju, A. Veerapathiran, V. K. Alagan, and K. Moorthy is acknowledged. The assistance of V. Rajamannar in bibliographical search is appreciated. Shri A. Venkatesh rendered help in the preparation of manuscript. We thank three anonymous reviewers for valuable comments and suggestions for improving the manuscript. References Cited Banerjee, K., P.V.M. Mahadev, M. A. Ilkal, A. C. Mishra, V. Dhanda, G. B. Modi, G. Geevarghese, H. N. Kaul, P. S. Shetty, and P. J. George. 1979. Isolation of Japanese encephalitis virus from mosquitoes collected in Bankura District (West-Bengal) during October 1974 to December 1975. Indian J. Med. Res. 69: 201Ð205. Chiang, C. L., and W. C. Reeves. 1962. Statistical estimation of virus infection rates in mosquito vector populations. Am. J. Hyg. 75: 377Ð391. Dhanda, V., and H. N. Kaul. 1980. Mosquito vectors of Japanese encephalitis virus and their bionomics in India. Proc. Indian Nat. Sci. Acad. India 46B: 759Ð768. Dhanda, V., V. Thenmozhi, N. P. Kumar, J. Hiriyan, N. Arunachalam, A. Balasubramanian, A. Ilango, and A. Gajanana. 1997. Virus isolation from wild-caught mosquitoes during a Japanese encephalitis outbreak in Kerala in 1996. Indian J. Med. Res. 106: 4 Ð 6. Gajanana, A., R. Rajendran, P. Philip Samuel, V. Thenmozhi, T. F. Sai, J. Kimura-Kuroda, and R. Reuben. 1997. Japanese encephalitis in South Arcot district, Tamil Nadu, India: a three-year longitudinal study of vector abundance and infection frequency. J. Med. Entomol. 34: 651Ð 659. Geevarghese, G., A. C. Mishra, and P. George Jacob, H. R. Bhat. 1994. Studies on the mosquito vectors of Japanese encephalitis virus in Mandya district, Karnataka, India. Southeast Asian J. Trop. Med. Public Health 25: 378Ð382. Geevarghese, G., P. C. Kanojia, and A. C. Mishra. 2004. Japanese encephalitis: vector ecology. In A. C. Mishra [ed.], National Institute of Virology Commemorative Compendium, Pune, India. Kabilan, L., R. Rajendran, and N. Arunachalam, S. Ramesh, S. Srinivasan, P. Philip Samuel, and A. P. Dash. 2004. Japanese encephalitis in India: an overview. Indian J. Pediatr. 71: 609 Ð 615. Downloaded from https://academic.oup.com/jme/article/46/3/633/861274 by guest on 13 February 2022 Cx. tritaeniorhynchus due to availability of perennial larval habitats such as semipermanent pools, riverbed pools created by rain, which persist throughout the year because of urban efßuents. Our entomological assessment indicated that Cx. tritaeniorhynchus was the primary vector based on relative abundance, and more number of virus isolations. Vector competence of Cx. tritaeniorhynchus was well demonstrated in laboratory studies (Mourya et al. 1991). Cx. tritaeniorhynchus has been incriminated as the principal vector of JE in many parts of India (Dhanda et al. 1997; Gajanana et al. 1997). The MIR of Cx. tritaeniorhynchus (0.63) was higher in Kurnool compared with MIR of Cx. tritaeniorhynchus (0.28) in Cuddalore, India (Gajanana et al. 1997). JEV infection in Cx. gelidus was observed only from periurban areas. JEV isolations also have been made from Cx. gelidus in India (Banerjee et al. 1979, Dhanda and Kaul 1980, Gajanana et al. 1997). MIR of Cx. gelidus (0.57) was higher in Kurnool compared with the MIR of Cx. gelidus (0.52) in Cuddalore, India (Gajanana et al. 1997). MIR and maximum likelihood estimates of JEV infections of Cx. gelidus were lower compared with the values obtained for Cx. tritaeniorhynchus, which indicate the primary role played by Cx. tritaeniorhynchus. Cx. gelidus is highly zoophagic and poorly anthrophagic; therefore, it may have an important role in amplifying JEV transmission (Reuben et al. 1992, Geevarghese et al. 1994). The seasonality of JEV transmission depends on various factors, among which the relative abundance of the vector species is important (Pant 1979). Our entomological investigations have improved our understanding of the seasonal patterns of JEV transmission which is highly seasonal in AP. In Kurnool, JE epidemic season begins in August, peaks in November, and then it declines dramatically in December each year as per state health records. The results indicated that Cx. tritaeniorhynchus abundance was highest during JE epidemic months, and JEV infections in vector mosquitoes also were found only during these months. JE is basically a rural disease because of the major JE vectors breed in rice Þelds. It is estimated that ⬇1.9 billion people currently live in rural JE-prone areas of the world. Among them, 220 million people live in proximity to rice irrigation schemes (Keiser et al. 2005). Pigs can become infected and act as amplifying hosts, bringing the virus closer to human habitats, especially in parts of Asia where pigs are kept near homes (Solomon 2006). In AP, pig rearing is very common among the weaker section of the communities, and there were 570 million pigs available as per May 2009 ARUNACHALAM ET AL.: JE VIRUS INFECTION IN VECTOR MOSQUITOES Reuben, R. Thenmozhi, V. Samuel, P. P. Gajanana, A., and T. R. Mani. 1992. Mosquito blood feeding patterns as a factor in the epidemiology of Japanese encephalitis in southern India. Am. J. Trop. Med. Hyg. 46: 654 Ð 663. Solomon, T. 2006. Control of Japanese encephalitisÑwithin our grasp? N. Engl. J. Med. 355: 869 Ð 871. Work, T. H., and K. V. Shah. 1956. Serological diagnosis of Japanese B type encephalitis in North Arcot District of Madras State, India, with epidemiological notes. Indian J. Med. Res. 10: 582Ð592. [WHO] World Health Organization. 2005. Global Advisory Committee on vaccine safety, 9Ð10. Wkly. Epidemiol. Rec. 80: 242Ð243. [WHO] World Health Organization. 2006. Out break encephalitis 2005: cases of Japanese encephalitis: Gorakhpur, Uttar Pradesh, India. 2005. Core Programme Clusters. Communicable Disease Surveillance. World Health Organization, Geneva, Switzerland. Received 7 May 2007; accepted 20 October 2007. Downloaded from https://academic.oup.com/jme/article/46/3/633/861274 by guest on 13 February 2022 Keiser, J., M. F. Maltese, T. E. Erlanger, R. Bos, M. Tanner, B. H. Singer, and J. Utzinger. 2005. Effect of irrigated rice agriculture on Japanese encephalitis, including challenges and opportunities for integrated vector management. Acta Trop. 95: 40 Ð57. Kimura-Kuroda, J., and K. Yasuri. 1983. Topographical analysis of antigenic determinants on envelope glycoprotein V3 (E) of Japanese encephalitis virus, using monoclonal antibodies. J. Virol. 45: 124 Ð132. Lacey, L. A., and C. M. Lacey. 1990. The medical importance of riceland mosquitoes and their control using alternatives to chemical insecticides. J. Am. Mosq. Control Assoc. 6 (Suppl.): 1Ð93. Mourya, D. T., A. C. Mishra, and R. S. Soman. 1991. Transmission of Japanese encephalitis virus in Culex pseudovishnui and Culex tritaeniorhynchus mosquitoes India. Indian J. Med. Res. 93: 250 Ð252. Pant, C. P. 1979. Vectors of Japanese encephalitis and their bionomics. WHO/VBC/79.732. World Health Organization, Geneva, Switzerland. Reuben, R., and A. Gajanana. 1997. Japanese encephalitis in India. Indian J. Pediatr. 64: 243Ð251. 639