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Rapid detection of Japanese encaphalitis virus by immunofluorescence after intracerebral inoculation of mosquito larvae
580 TRANSACTIONSOF THE ROYAL SOCIETYOP TROPICALMEDICINE AND HYGIENE(1990) 84, 580
1 Short Report 1 Rapid detection of Japanese encaphalitis virus by immunofluorescence after intracerebral inoculation of mosquito larvae D. T. Mourya Division of Medical Entomology and Zoology, National Institute of Virology, 20-A Dr Ambedkar Road, Pune 411 001, India The rapid detection of dengue viruses by immunofluorescence following intracerebral inoculation of Toxorhynchychites splendens larvae and adults was first reported by PANGet al. (1983) and THET-WIN (1982). Immunofluorescence was detected from the 2nd day after infection onwards in larvae and from the 5th day after infection onwards in adult mosquitoes. The use of this technique for detection of the multiplication of an Indian strain (P-20778) of Japanese encephalitis virus (JEV) in TX. splendens, third to fourth stagelarvae and adults, is reported for the first time. The virus strain, originally isolated from human brain at Vellore, India, was used at the fifteenth mouse passage. The titre of the stock suspension was 7.5 dex (mouse, intracerebral) per 0.02 ml. Serial ten-fold dilutions ranging from 10-l to lo-’ were inoculated. The inoculated-mosquitoes were incubated at 28”Ctl”C and 80%&S% relative humidity. JEV was detected in head-squeeze smears of the larvae 24 h after infection, even when inoculated at a dilution of 10e4,and on the third day using a dilution of 10e5. Larvae inoculated with the end dilution, lo-*, showed antigen after 4-S d. In adult mosquitoes inoculated intracerebrally, JEV was detected 24 h after inoculation using dilutions u to 10e3, and on the 3rd day using a dilution of lo- P. In comparison, adults infected by intrathor-
acic inoculation (ROSEN & GUBLER, 1974) were negative on the 1st and 2nd days after inoculation, becoming positive only on the 3rd day, using dilutioni of 10-l and lo-’ onlv. Mortalitv was low exceot in adults inoculated intracerebrally; when it was about 40%.
A significant improvement in the technique used to inoculate larvae was to introduce the needle through the soft membranous junction at the dorso-posterior end of the head capsule, into the oesophagealganglion, instead of dorsally through the epistome. Both methods were equally sensitive but larval mortality was reduced to a negligible level with the improved method compared to approximately 20% with the former method. When the comparative susceptibility of larvae of Culex bitaeniorhynchus, C. tritaeniorhynchus and Aedes aegypti to JEV was studied by intracerebral inoculation, the larvae of both the Culex specieswere found to be as susceptible as TX. splendens larvae. However, Aedes larvae were slightly less sensitive and virus could be detected up to 10e3 dilution on the 1st day after infection and up to 10v4 on the 2nd and 3rd days. PANGet al. (1983) suggested that there is rapid division of brain cells in the larval stage, which supports the rapid replication of the virus. The detection of viral antigen in the brain cells as early as the first day after inoculation of such low dilutions suggests that the replication of JEV is much faster than that of dengue viruses. This may be due to the fact that JEV is a neurotropic virus. This system has considerable potential for the rapid detection of JE virus. References Pang, T., Lam, S. K., Chew, C. B., Poon, G.
K. & Ramalingam,S. (1983).Detectionof denguevirus by immunofluorescence following inoculation of mosquito
larvae. Lancet, i, 1271.
Rosen,L. & Gubler, D. (1974).The useof mosouitoesto deiectandpropagated&g&viruses. Americanjournal of Tropical Medicine and Hygiene, 23, 11534-1160. Thet-win (1982). Detection of dengue virus by immunofluorescence afterintracerebralinoculationof mosquitoes. Lancet, i, 53-54. Received 20 December 1989; revised 6 February 1990; accepted for publication 6 February 1990
1 Short Report 1 Rapid detection of Japanese encaphalitis virus by immunofluorescence after intracerebral inoculation of mosquito larvae D. T. Mourya Division of Medical Entomology and Zoology, National Institute of Virology, 20-A Dr Ambedkar Road, Pune 411 001, India The rapid detection of dengue viruses by immunofluorescence following intracerebral inoculation of Toxorhynchychites splendens larvae and adults was first reported by PANGet al. (1983) and THET-WIN (1982). Immunofluorescence was detected from the 2nd day after infection onwards in larvae and from the 5th day after infection onwards in adult mosquitoes. The use of this technique for detection of the multiplication of an Indian strain (P-20778) of Japanese encephalitis virus (JEV) in TX. splendens, third to fourth stagelarvae and adults, is reported for the first time. The virus strain, originally isolated from human brain at Vellore, India, was used at the fifteenth mouse passage. The titre of the stock suspension was 7.5 dex (mouse, intracerebral) per 0.02 ml. Serial ten-fold dilutions ranging from 10-l to lo-’ were inoculated. The inoculated-mosquitoes were incubated at 28”Ctl”C and 80%&S% relative humidity. JEV was detected in head-squeeze smears of the larvae 24 h after infection, even when inoculated at a dilution of 10e4,and on the third day using a dilution of 10e5. Larvae inoculated with the end dilution, lo-*, showed antigen after 4-S d. In adult mosquitoes inoculated intracerebrally, JEV was detected 24 h after inoculation using dilutions u to 10e3, and on the 3rd day using a dilution of lo- P. In comparison, adults infected by intrathor-
acic inoculation (ROSEN & GUBLER, 1974) were negative on the 1st and 2nd days after inoculation, becoming positive only on the 3rd day, using dilutioni of 10-l and lo-’ onlv. Mortalitv was low exceot in adults inoculated intracerebrally; when it was about 40%.
A significant improvement in the technique used to inoculate larvae was to introduce the needle through the soft membranous junction at the dorso-posterior end of the head capsule, into the oesophagealganglion, instead of dorsally through the epistome. Both methods were equally sensitive but larval mortality was reduced to a negligible level with the improved method compared to approximately 20% with the former method. When the comparative susceptibility of larvae of Culex bitaeniorhynchus, C. tritaeniorhynchus and Aedes aegypti to JEV was studied by intracerebral inoculation, the larvae of both the Culex specieswere found to be as susceptible as TX. splendens larvae. However, Aedes larvae were slightly less sensitive and virus could be detected up to 10e3 dilution on the 1st day after infection and up to 10v4 on the 2nd and 3rd days. PANGet al. (1983) suggested that there is rapid division of brain cells in the larval stage, which supports the rapid replication of the virus. The detection of viral antigen in the brain cells as early as the first day after inoculation of such low dilutions suggests that the replication of JEV is much faster than that of dengue viruses. This may be due to the fact that JEV is a neurotropic virus. This system has considerable potential for the rapid detection of JE virus. References Pang, T., Lam, S. K., Chew, C. B., Poon, G.
K. & Ramalingam,S. (1983).Detectionof denguevirus by immunofluorescence following inoculation of mosquito
larvae. Lancet, i, 1271.
Rosen,L. & Gubler, D. (1974).The useof mosouitoesto deiectandpropagated&g&viruses. Americanjournal of Tropical Medicine and Hygiene, 23, 11534-1160. Thet-win (1982). Detection of dengue virus by immunofluorescence afterintracerebralinoculationof mosquitoes. Lancet, i, 53-54. Received 20 December 1989; revised 6 February 1990; accepted for publication 6 February 1990