Failure of Dengue Viruses To Replicate in Culex quinquefasciatus (Diptera: Culicidae) G. HUANG, E. VERGNE, AND D. J. GUBLER Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control, Public Health Service, U.S. Department of Health & Human Services, P.O. Box 2087, Fort Collins, CO 80522

J. Med. Entomol. 29(6): 911-914 (1992)

KEY WORDS dengue, mosquitoes, virus replication

I N RECENT YEARS, several investigators in China and Vietnam have reported the isolation of

dengue viruses from Culex quinquefasciatus Say collected in nature (Chen et al. 1982, 1986; Do Quang Ha, Pasteur Institute, Ho Chi Minh City, Vietnam, personal communication). Moreover, results of experimental studies carried out in China suggest that this species not only became infected, but that it was capable of oral transmission of dengue viruses to mice (Liu & Zhao 1985, Tong et al. 1987). These results are in contrast to previously published experimental studies of dengue viruses in Cx. quinquefasciatus. The purpose of our study was to determine whether New World Cx. quinquefasciatus were capable of becoming infected with and supporting the replication of dengue viruses. Materials and Methods Viruses. The dengue viruses used in these experiments were the prototype strains (DEN-1, Hawaii; DEN-2, New Guinea C; DEN-3, H87; and DEN-4, H241). All had been passaged several times in mosquitoes but none had been passed in mice or cell cultures. Stock pools of virus were prepared by intrathoracic inoculation of Toxorhynchites amboinensis (Dohschall), using the method of Rosen & Gubler (1974). After incubation for 10 d at 30°C, mosquitoes were killed by freezing and triturated in phosphatebuffered saline (PBS), pH 7.4, containing 30% heat-inactivated calf serum (56°C for 30 min). Use of trade names is for identification only and does not imply endorsement by the Public Health Services or by the U.S. Department of Health and Human Services.

After centrifugation at 1,575 x g for 30 min at 5°C, aliquots of the supernatant fluid were dispensed into screw-cap vials and stored at -70°C. Mosquitoes. The Cx. quinquefasciatus mosquitoes used in these experiments were collected as eggs on the grounds of the San Juan Laboratories, Centers for Disease Control (CDC), San Juan, Puerto Rico. Eggs were hatched and reared in white enamel pans (25 by 41 cm) on a diet of Purina rabbit chow pellets (Purina Mills, St. Louis, MO) and Tetramin (Tetra Werke, West Germany) fish food. Pupae were picked and adults were allowed to emerge in cages (30 by 30 by 30 cm) in which they were provided a maintenance diet of 5% sucrose for 5-7 d before attempts were made to infect them. The Ae. aegypti and Tx. amboinensis were from colonies that had been maintained at the San Juan laboratories for several years. The Ae. aegypti originally were collected from Rexville, Puerto Rico, as larvae in 1982 and had been maintained in the laboratory since that time, with periodic infusion of larvae from Rexville over the years to maintain genetic variability. Susceptibility to infection with dengue viruses remained unchanged in this strain during this time (D.J.G., unpublished data). The Tx. amboinensis were from a colony begun with specimens collected on Oahu, HI, in 1975. These two species were used interchangeably for virus assay because there is no difference in their susceptibility to parenteral infection with dengue viruses (D.J.G., unpublished data). Infection and Assay of Mosquitoes. A total of 60 Cx. quinquefasciatus and 50 Ae. aegypti (3-7

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ABSTRACT Culex quinquefasciatus (Say) and Aedes aegypti (L.) were parenterally infected with dengue viruses and virus replication was monitored at intervals after infection in each species. Dengue viruses replicated rapidly in Ae. aegypti, reaching a peak titer of 10 6 -10 7 mosquito infectious dose 50 (MIDSQ) per mosquito. In Cx. quinquefasciatus, however, dengue virus replication did not occur. We conclude that this mosquito species is refractory to infection with dengue viruses and, therefore, does not serve as a vector in nature.

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D CO

O

X 0 1 2 3 I 5 6 7* 8 I 10 fi 12

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Days after Inoculation Fig. 1. Growth curves of DEN-1, DEN-2, DEN-3, and DEN-4 after parenteral infection of Ae. aegypti (*) and Cx. quinquefasciatus (X).

d old) was inoculated with 102 MIDgo of each of the four dengue serotypes using the method of Gubler & Rosen (1974). After inoculation, mosquitoes were incubated at 30°C and 65-75% relative humidity (RH) on a maintenance diet of 5% sucrose. On days 2,4, 7, 9, 11, and 15 after infection, five mosquitoes of each species were killed by freezing and individually assayed for virus (Gubler & Rosen 1977). Briefly, individual mosquitoes were triturated in 1.0 ml of PBS containing 30% heat-inactivated calf serum, centrifuged at 1,575 x g for 30 min at 5°C, and serial 10-fold dilutions of the supernatant fluid were inoculated into groups of uninfected Ae. aegypti or Tx. amboinensis. These mosquitoes were incubated for 10 d at 30°C and 65-75% RH, then individually examined for the presence or absence of viral antigen in head squashes by the direct fluorescent antibody (DFA) test (Kuberski & Rosen 1977) using a fluorescein isothiocyanate conjugate prepared from pooled human serum samples that had high (^5,120) flavivirus hemagglutination-inhibition antibody titers. Generally, at least five mosquitoes inoculated with each dilution were tested by DFA, and the MID 50 was calculated by the method of Reed & Muench (1938).

Another group of five mosquitoes (from those inoculated in the above experiment) were saved on the same days noted above and examined for disseminated virus infection using DFA to detect dengue virus antigen in head squashes. Results Dengue virus antigen was detected in head squashes of some Ae. aegypti by the fourth day after infection and in 100% of this species by day 10. Virus was not detected by DFA in the head squashes of any Cx. quinquefasciatus up to 35 d following parenteral infection. Moreover, dengue virus was not detected in individually triturated Cx. quinquefasciatus that had been inoculated with 102 MIDso of dengue virus and incubated for periods up to 35 d at 30°C (Fig. 1). In contrast, replication of the four prototype dengue viruses following parenteral infection of Ae. aegypti was very rapid (Fig. 1). Each point of the growth curves represents the geometric mean virus titer of at least five mosquitoes. The virus content observed in Ae. aegypti after parenteral infection was similar for all four serotypes. Replication of virus was detectable by day 2 and peaked on day 9 after infection (DEN-2

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DEN - 4

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and DEN-4) and day 11 after infection (DEN-1 tions from mosquitoes collected in nature, but and DEN-3). Maximum mean virus titers then not for the experimental results showing transbegan to decline. As noted above, none of the mission of dengue viruses to mice. Unfortufour prototype dengue viruses could be detected nately, we were not able to study Cx. quinquein Cx. quinquefasciatus on any day after infec- fasciatus strains from China and Vietnam; thus, tion. we cannot rule out this species as a dengue vector in those countries. As noted above, however, it is difficult to reconcile these striking differDiscussion ences in results. We conclude that Cx. quinqueOur results are identical to, and support those fasciatus is refractory to infection with dengue of, previous investigators who attempted to ex- viruses and is, therefore, not a vector for these perimentally infect Cx. quinquefasciatus with viruses in nature. dengue viruses, and they support the conclusion that this mosquito species is not a susceptible References Cited host for these viruses (Cleland et al. 1919, Siler et al. 1926, Snijders et al. 1931, Eshita 1982, Chen, W., L. Zhou, X. Liu, J. Wan, Z. Zhao, C. Lin, Y. Ji, Q. Cui & Y. Yu. 1982. Recovery of dengue-3 Rosen et al. 1985). Because dengue viruses do viruses from Culex fatigans during an epidemic of not replicate in Cx. quinquefasciatus, even after dengue fever in Hainan Island in 1981. Chin. J. parenteral infection, it is not likely that this speMicrobiol. Immunol. 2: 363-364. cies can serve as a vector for these viruses in Chen, W., Y. Chen, J. Kuang, Y. Liu, C. Wang, Z. Zhao, nature. F. Xie & J. Chen. 1986. An epidemic of dengue Experimental transmission studies and virus fever caused by dengue virus type 2. Chin. J. Miisolation results from field-collected Cx. quincrobiol. Immunol. 6: 204-206. quefasciatusfromChina and Vietnam, however, Cleland, J. B., B. Bradley & W. McDonald. 1919. contradict these results. A possible explanation Further experiments in the etiology of dengue fever. J. Hyg. 18: 217-254. for the conflicting results is that geographically diverse strains of Cx. quinquefasciatus may vary Eshita, Y. 1982. Experimental studies on the transmission of dengue by Japanese mosquitoes. Tokyo in their susceptibility to infection with dengue Med. J. 5: 17-27. viruses. Although geographic strain variation to Gubler, D. J. & L. Rosen. 1976. Variation among infection with dengue viruses has been docugeographic strains oi Aedes albopictus in susceptimented in mosquito vector species (Gubler & bility to infection with dengue viruses. Am. J. Trop. Rosen 1976, Gubler et al. 1979, Rosen et al. Med. Hyg. 25: 318-325. 1985), it has never been shown that some strains 1977. Quantitative aspects of replication of dengue of the same mosquito species are completely reviruses in Aedes albopictus (Diptera: Culicidae) after oral and parenteral infection. J. Med. Entomol. fractory to parenteral infection with dengue and 13: 469-472. others are susceptible. Moreover, four geographically diverse strains of Cx. quinquefaciatus, two Cubler, D. J., S. Nalim, R. Tan, H. Saipan & J. SuliantiSaroso. 1979. Variation in susceptibility to oral from Asia (Philippines [Siler et al. 1926]; Japan infection with dengue viruses among geographic [Eshita 1982]), one from the Pacific (Hawaii strains of Aedes aegypti. Am. J. Trop. Med. Hyg. 28: [Rosen et al. 1985]), and one from the Americas 1045-1052. (Puerto Rico) showed the same results in care- Kuberski, T. T. & L. Rosen. 1977. A simple techfully controlled experiments, indicating that innique for the detection of dengue antigen in mosterstrain variation is probably not the explanaquitoes by immunofluorescence. Am. J. Trop. Med. Hyg. 26: 533-537. tion. This conclusion is supported by the fact that when geographic variation in susceptibility to Liu, X. & G. Zhao. 1985. Studies on the experimental transmission of dengue virus by Culex fatigans. arbovirus infection is observed in strains of mosChin. J. Microbiol. Immunol. 5: 247-249. quitoes, resistance to infection usually can be overcome by bypassing the midgut barrier Reed, L. J. & H. Muench. 1938. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27: (Gubler & Rosen 1976). This was not the case 493-497. with Cx. quinquefasciatus and dengue viruses. Rosen, L. & D. J. Gubler. 1974. The use of mosquiAlternative explanations include problems in toes to detect and propagate dengue viruses. Am. J. laboratory technique and the possibility that old Trop. Med. Hyg. 23: 1153-1160. Ae. aegypti (variety queenslandensis Theobald), Rosen, L., L. E. Roseboom, D. J. Gubler, J. C. Lien & B. N. Chaniotis. 1985. Comparative susceptibilwith scales rubbed off and nearly brown in color, ity of mosquito species and strains to oral and were mistakenly identified and mixed with pools parenteral infection with dengue and Japanese enof Cx. quinquefasciatus. This, in fact, is the excephalitis viruses. Am. J. Trop. Med. Hyg. 34: 603planation given for the conflicting results of early 615. investigators (Siler et al. 1926). Another exJ. F., M. W. Hall & A. P. Hitchens. 1926. planation is that a leg broken off from an infected Siler, Dengue, its history, epidemiology, mechanism of Ae. aegypti could adhere to an uninfected Cx. transmission, etiology, clinical manifestations, imquinquefasciatus during pooling. These explamunity and prevention. Phil. J. Sci. 29: 1-304. nations could account for the dengue virus isola- Snijders, E. P., E. J. Dinger & W.A.P. Schuffuner.

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1931. On the transmission of dengue in Sumatra. Am. J. Trop. Med. Hyg. 11: 171-197. Tong, S., Q. Zang, G. Li, P. Zhang, T. Wang & J. Zhang. 1987. Experimental study on vector competence of several species of mosquitoes of China

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with dengue virus. J. Acad. Mil. Med. Sci. 11: 458462. Received for publication 23 October 1991; accepted 30 March 1992.

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Failure of dengue viruses to replicate in Culex quinquefasciatus (Diptera: Culicidae).

Culex quinquefasciatus (Say) and Aedes aegypti (L.) were parenterally infected with dengue viruses and virus replication was monitored at intervals af...
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