MULTIPLICATION OF DENGUE AND CHIKUNGUNYA VIRUSES IN AEDES MOSQUITOES

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1 MULTIPLICATION OF DENGUE AND CHIKUNGUNYA VIRUSES IN AEDES MOSQUITOES Soedarto ~oekiman* ABSTRACT Colonies of Aedes aegypti (Surabaya strain) and Aedes albopictus (Malang strain) were studied to compare their susceptibility to oral infection with dengue type 3 and chikungunya viruses. Growth curves of dengue type 3 and chikungunya viruses in these mosquitoes indicated that both mosquito species were susceptible to oral infection with these viruses. Electron microscopic observation of the salivary glands of A. aegypti and A. albopictus infected with chikungunya virus showed that this organ plays an important role in producing and maintaining high virus titers in these mosquitoes. The results suggest that both Aedes species are potentially important vectors on the transmission of dengue and chikungunya infection in Indonesia. INTRODUCTION Dengue hemorrhagic fever is still a major health problem in Indonesia. In 1968, 58 DHF cases with 41.3 % Case Fatality Rate (CFR) were reported from Indonesia, while in 1983 the number of cases were increased to 13,875 with 3.8 % CFR. Primarily DHF was recognized as one of the urban diseases, of which most cases were reported from larger cities in Java Island such as Surabaya and Jakarta. Since 1980 DHF epidemics have spread centrifugally to smaller urban and rural communities in all provinces of Indonesia except Timor-Timur (East Timor). In Indonesia all four types of dengue viruses have been recovered from patients with DHF, of which dengue type 3 virus was the re J$.j dominant virus isolated from their sera. Seroepidemiological surveillance revealed that chikungunya virus, an alphavirus, was also distributed in Indonesia4. This virus causes a disease clinically resembling the haemorrhagic fever. Aedes aegypti and Aedes albopictus are common mosquito species in Indonesia. A. aegypti is a domestic mosquito widely found indoors, while A. albopictus is widely distributed outdoors in rural and urban In most parts of the world the principle vector of dengue viruses is A. aegypti mosquito, but in Japan, the Seychelles in Indian ocean, and some other countries A. albopictus is the sole 7 vector. Virus isolates can be made from acute sera, plasma, leucocytes or organs of DI-IF patients. The material is inoculated preferably into mosquitoes, mosquito cell culture or vertebrate culture cells such as LLC-MK2 (Rhesus Monkey Kidney Cell) and BHK-21 (Baby Hamster Kidney Cell) cell lines. Toxorhynchites (TRA 284) and Aedes (C6/ * Department of Parasitology, Medical Faculty Airlangga University Surabaya, Indonesia Bul. Penelit. Kesehat. 17 (2) 1989

2 36) cell lines are most commonly used for the primary isolation of dengue viruses. These cells are not easily available in this country. The other method for dengue and chikungunya isolation is by direct inoculation of mosquitoes by intrathoracal injection or by feeding method. Dengue and chikungunya antigens can be detected by positive immunofluorescence using polyclonal or monoclonal antibody in the direct or indirect immunofluorescent antibody (IFA) testsq9. There are wide variations in susceptibility to experimental infection with dengue and chikungunya viruses among different species of mosquitoes and different strains of the same species 2*10y1 '*12. Biological and ecological similarities have been indicated between A. aegypti and A. albopictus collected in East ~ava'~. For the elucidation of the virus susceptibility, the multiplication of dengue and chikungunya viruses in A. aegypti and A. albopictus mosquitoes after feeding on bloodvirus mixture was studied. MATERIALS AND METHODS Mosquitoes Colonies of A. aegypti (Surabaya strain) and A. albopictus (Malang strain) were mainly used. Mosquitoes were reared in an insectary maintained at 27'~, with % relative humidity and 16:8 (L:D) photoperiod. Larva were given a diet of mouse pellets, and adult mosquitoes were provided with 0.7 % sucrose solution. Viruses Dengue type 3 virus, H87 strain, was provided from the Department of Microbiology Kobe University, at the 32th suckling mouse brain passage. The seed virus was obtained from 10 % homogenate of infected brain specimens in phosphate-buffered saline (PBS). An African strain of chikungunya virus was also used,,in which the seed virus was the culture fluid obtained from infected BHK-21 monolayer cultures 14. Infection of mosquitoes For infection of mosquitoes with dengue type 3 and chikungunya viruses, 4 to 10-dayold female mosquitoes were allowed to feed on virus defibrinated sheep blood mixtures with an appropriate titer of infective virus. At 1 to 2-day intervals, 5 to 12 mosquitoes of each species were frozen at -80'~ until used for viral assay. Virus assay Each pool of mosquitoes was homogenized in 2 ml of culture medium (Eagle's minimum essential medium supplemented with 0.03 % sodium bicarbonate, 5 % heatinactivated calf serum and 0.03 % L- glutamine), cenntrifuged at 700xg for 5 minutes and then filtered through a Minisart NP membrane with 0.45 pm porosity. The filtrate was titrated for viral infectivity by the plaque assay method14 or by the indirect fluorescent antibody technique essentially based on the original focuscounting method8. The titer was expressed as plague forming unit (PFU) or focus forming unit (m). RESULTS Growth of dengue type 3 virus in Aedes mos uitoes was studied after feeding on virus 977 at 10 ' FFU per ml. As shown in Table 1, the virus titer on day 0 (the datum immedi- BuL Penelit. Kesehat. 17 (2) 1989

3 ately after feeding) was 10"~ FFW per ml in both species. On day 2 the titers dropped to 10~.~ FFU in Ae. aegypti and FFU in Ae. albopictus. The titer of dengue type3 virus reached a maximum of 10~'~ FFU in Ae. aegypti and 10'" PN in Ae. albopictus both on day 12. Growth of chikungunya virus in Ae. aegypti and Ae. albopictus is shown in table 2. In this experiment, mosquitoes received an infective meal with a titer of 10'" PFU per ml. On day 0 the virus titers were PFU in Ae. aibopictus and 103.' PFU in Ae. aegypti. On day 2 the titers dropped to lo3'' PN in Ae. albopictus and PN in Ae. aegypti. After that, the titers started to increase in both species and reached the maximum titers of lo5e4 PFU on day 13 in Ae. albopictus and of 1 o ~ PN. on ~ day 10 in Ae. aegypti. The virus titer in Ae. aegypti then dropped quickly and became undetectable on day 20. On the other hand, the virus titer in Ae. albopictus declined slowly to PFU on day 20. DISCUSSION. Pattern of dengue type 3 virus multiplication in Ae. aegypti showed that the virus titer on day 2 was one fifth of the titer on day 0, while inae. albopictus the virus titer on day 2 was 100 times as low as that on day 0. On the other hand, the initial drop was remarkable in chikungunya virus - infected Ae. aegypti in which the titer on day 2 was 5,000 times lower than the virus titer on day 0. The initial drop observed in this study was consistent with other reports2*16 reporting that on the 2nd day of oral infection, no chikungunya virus was recovered from hemolymph of Ae. aegypti mosquitoes. Presence of virus antigen was restricted in the posterior midgut cells of Ae. albopictus infected with dengue type 2 virus as reported by ~uberski". These results suggest that a "gut barrier" was present in the midgut of mosquitoes infected with virus by oral feeding. Titers of chikungunya and dengue type 3 viruses in infected mosquitoes increased after the second day of infection. In Ae. aegypti infected with dengue type 3 virus, the maximum titer was 4000 times as high as the titer on day 0, while in Ae. albopicfzu the maximum titer was 800 times higher than the titer on day 0. In Ae. aegypti and Ae. albopictus mosquitoes infected with chikungunya virus, the maximum titers were 8 times and 60 times as high as the titer on day 0, respectively. Aftemaching the maximum, the titer began to decrease on the next day, indicating a limited period of efficient virus multiplication in the mosquito body. The maximum titers of chikungunya virus in the Surabaya strain of Ae. aegypti and the Malang strain of Ae. albopictus were almost of the same magnitude in both Aedes species. Differences in some experimental conditions may affect the virus multiplication in these mosquitoes. High dengue type 3 virus titers observed in the present study using Ae. aegypti and Ae. albopictus were similar to those reported by Gubler and Rosen (1976)'' and Gubler et al. (1979~)~ with several other geographic strains. Electron microscopic observations on salivary glands of Ae. aegypti and Ae. albopictus infected with chikungunya virus by oral feeding suggested that the salivary glands of' these mosquitoes play an important role in producing and maintaining a high titer of chikungun a virus, similar as in the case of?8 dengue virus. It is concluded from these experimental, biological and electron microscopic observations that these two Aedes species serve as potent vectors of dengue type 3 and chikungunya viruses. Especially, Ae. aegypti is im- Bul. Penelit. Kesehat. 17 (2) 1989

4 Table 1. Titer of dengue type 3 virus in Aedes mosquitoes after feeding on virus at 10~'~ FFU per ml* Days Post- Titer of virus in Log (FFU/mosquito) infection Aedes albopictus Aedes oegypti - -- * Mean virus titers obtained from 5-12 mosquitoes per pool, were represented as FFU per mosquito Table 2. Titer of chikungunya virus in Ae&s mosquitoes after feeding on virus at PFU per ml* Days post infection Titer of virus in Log (PFU/mosquito Aedes albopictus Aedes aegypti * Mean virus titer obtained from 5-12 mosquitoes per pool, was represented as PFU per mosquito. Bul. Penelit. Kesehat. 17 (2) 198

5 portant in urban areas and Ae. albopictus in rural areas. Studies on replication of dengue and chikungunya viruses in Aedes mosquitoes should be done more intensively to evaluate the potential vector competence of these mosquito species and strain and also may be relevant in characterizing attenuated strains of dengue and chikungunya viruses with respect to their suitability for use in vaccines. ACKNOWLEDGEMENT The author wishes to thank Professor Takeo Matsumura, Eiji Konishi, PhD, the staff of the Department of Medical Zoology, Professor Mono Homma from the Department of Microbiology, Professor Hiroshi Itoh, Department of Pathology, Kobe University School of Medicine and Professor Hiroshi Yarnanishi, Kobe Gakuin Women's Junior College for the efforts to completion of this study. This study was performed as a part of the Ronpaku Doc torate Program in Kobe University, partly supported by Japan Society for the Promotion of Science and International Center for Medical Research Kobe University. REFERENCES 1. Sunoto, P.F.D. Van Peenen, Sumarmo, Samo Sinto. and P.L. Joseph (1975). Studies on dengue in. children at Dr. Cipto Mangunkusumo General Hospital, Jakarta (May 1973-January 1974). Paed. Indonesiana. (15): Gubler. D.J., S. Nalim, R. Td, H. Saipan, and J.S. Saroso (1979a). Variation in susceptibility to oral infection with dengue viruses among geographic strains of Aedes aegypti. Am.J. Trop.Med.Hyg. (28): Gubler, D.J.. W. Suharjono, I. Lubis, S. Eram, and J.S. Saroso (1979b). Epidemic dengue hemorrhagic fever in rural Indonesia. I. Virological and epidemiological studies. Am. J.Trop.Med.Hyg. (28): Hotta, S. (1978). Dengue and related tropical viruses. Yukosha Printing House, Kobe, Japan. 5. Oda, T.. A. Igarashi. S. Hotta, N. Fujita, Y. Funahara. S. Djakaria, R. Hudoyo, A. Isfarain, and D. Djohor (1984). Studies on bionomics of Aedes aegypti and Aedes albopictus and dengue virus isolation in Jakarta, Indonesia. ICMR Annals. (3): Jumali, Sunarto, D.J. Gubler, S. Nalim, S. &am, and J.S. Saroso (1979). Epidemic dengue hemorhagic fever in rural Indonesia. III. Fhtomological studies. Am.J.Trop.Med.Hyg. (28): Metselaar, D., C.R. Grainger, K.G. Oei, D.G. Reynolds, M. Pudney, C.J. Leake, P.M. Tukei, R.M. D'Offay, and D.I.H. Simpson (1980). An outbreak of type 2 dengue fever in the Seychelles, probably transmitted by Aedes albopictus (Skuae). Bull.WWO (58): Igarhasi. A., and M. Mantani (1974). Rapid titration of dengue virus type 4 infectivity by counting fluorescent foci. Biken J. (17): World Health Organization (1985). Viral Haemorrhagic Fevers. WHO Toch.Rep.Ser Gubler. DJ.. and L. Rosen (1976). Variation among geographic strains of Aedes albopictus in susceptibility to infection with dengue viruses. Am.J.Trop.Med.Hyg. (25): Tesh, R.B., D.J. Gubler, and L. Rosen (1976). Variation among geographic strains of Aedes albopictus in ausceptibiity to infection with chikungunya virus. Am.J.Trop.Med.Hyg. (25): Eshita, Y. (1982). Experimental studies on the transmission of dengue virus by Japanese mosquitoes. Teikyo Medical Journal (5): Soekiman, S., Machfudz, Subagyo, S. Adipoetro, H. Yamanishi, and T. Matsumura (1984). Comparative studies on the biology of Aedes negypti (Linnaeus, 1762) and Aedes albopictus (Skuse, 1895). ICMR Annals. (4): Konishi, E., and S. Hotta (1979). Effects of tannic acid and its related compounds upon chikungunya virus. Microbial. Immunol, (23):

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