Susceptibility of Selected Strains of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) to Chikungunya Virus MICHAEL J. TURELL, JOSEPH R. BEAMAN, AND RALPH F. TAMMARIELLO Virology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21702
J. Med Entomol. 29(1): 49-53 (1992) ABSTRACT The relative susceptibility of selected strains of Aedes aegypti (L.) and Aedes albopictus (Skuse) fed on a viremic monkey to infection with chikungunya virus was determined. Infection rates were consistently higher in 10 strains of Ae. albopictus tested than in 7 strains of Ae. aegypti tested, regardless of the geographic location from which the strains originated or the dose of virus ingested. Similarly, virus dissemination rates were higher in the Ae. albopictus strains compared with the Ae. aegypti strains. For nearly all (11 of 12) strains tested of both species, groups of mosquitoes with one or more females with a disseminated infection transmitted virus by bite to weanling mice. Based on these studies, Ae. albopictus appears to be a more competent laboratory vector of chikungunya virus than does Ae. aegypti. KEY WORDS Insecta, Aedes spp., chikungunya virus, infection rates
Aedes aegypti (L.) is considered to be the principal vector of chikungunya (CHIK) virus in Asia, various studies indicate that this species has a high threshold of infection for CHIK virus (Jupp & Mclntosh 1988). Other studies have indicated that Aedes albopictus (Skuse) may be a potential secondary vector for this virus (Mangiafico 1971, Jupp & Mclntosh 1988). The recent introduction of this species into the Americas has raised concern that it may serve as a vector for exotic as well as endemic viruses (Knudson 1986). This species is a competent laboratory vector for numerous arboviruses (Shroyer 1986, Hawley 1988). In addition, Ae. albopictus can displace populations of Ae. aegypti in the southern United States (J. Freier, personal communication) and has demonstrated an ability to flourish in tree holes as well as in artificial containers (Hawley 1988). Because the potential for Ae. albopictus (Tesh et al. 1976) and Ae. aegypti (Banerjee et al. 1988) to transmit CHIK virus differs for strains isolated from different geographic locations, we evaluated 10 strains of Ae. albopictus and 7 strains of Ae. aegypti for their ability to transmit CHIK virus. Most previous studies on the ability of Aedes mosquitoes to transmit CHIK virus have used techniques such as droplet feeding, bloodsoaked pledgets, or membrane feeders to expose mosquitoes to virus. Virus infection rates in mosquitoes that are artificially exposed to virus may be significantly lower than in mosquitoes that ingested the same virus dose from a viremic animal (Jupp 1976, Meyer et al. 1983, Miller 1987, Turell 1988). Therefore, to use a more natural system and to ensure that mosquitoes were exALTHOUGH
posed to the same virus dose, we allowed up to 10 different strains of mosquitoes to feed concurrently on the same viremic rhesus monkey. Materials and Methods Mosquitoes. Ten geographic strains of Ae. albopictus and seven of Ae. aegypti were obtained from various sources (Table 1). With the exception of two strains that had been colonized for many years, attempts were made to use newly established colonies as much as possible. All strains were maintained at 26°C and high humidity according to procedures described by Gargan et al. (1983). Female mosquitoes were between 4 and 10 d old when used for infection trials. Virus and Virus Assay. Strain 15561 of CHIK virus was isolated from human serum in Thailand during the 1962 epidemic. It was passaged twice in primary green monkey kidney cells before its use in these studies (Levitt et al. 1986). Serial 10-fold dilutions of specimens were tested for infectious virus by plaque assay on Vero cell monolayers. Determination of Vector Susceptibility. Between 50 and 70 female mosquitoes from each strain were placed in 0.5-liter cardboard containers (one strain per container) with netting over one end. To ensure a similar virus exposure for each strain tested, mosquitoes in up to 10 of these containers were allowed to feed concurrently on each of three anesthetized rhesus monkeys that had been inoculated subcutaneously 24 or 48 h earlier with 0.2 ml of a suspension containing 107-108 plaque-forming units of CHIK virus. There were five feedings (two at 24 h and
JOURNAL OF MEDICAL ENTOMOLOGY
50
Vol. 29, no. 1
Table 1. Strains of Ae. aegypti and Ae. Albopictus Species
Strain
Collection location (yr collected)
Ae. albopictus
GENTILLY HOUSTON MADAGASCAR OAHU OKINAWA POLK SABAH SAO PAULO TAIWAN ZAMA LAS VIRTUDES REX ROCKEFELLER THAILAND GENTILLY FOSTER DAKAR
New Orleans, La. (1988) Houston, Tex. (1985) Antananarivo, Madagascar (1987) Honolulu, Hawaii (1971) Naha, Okinawa (1987) Polk, Fl. (1989) Sabah, Malaysia (1986) Sao Paulo, Sao Paulo, Brazil (1987) Kaohsiung, Taiwan (1989) Tokyo, Japan (1986) San Juan, Puerto Rico (1988) Puerto Rico (1987)
Ae. aegypti
Generation
Bangkok, Thailand (1989) New Orleans, La. (1988) Columbus, Ind. (1986) Dakar, Senegal (1988)
three at 48 h after inoculation with virus). Before each feeding attempt, blood was removed from the femoral vein to determine the viremia level. In addition, immediately after several of the feedings, six engorged mosquitoes were individually triturated in 1 ml of diluent (10% fetal bovine serum in Medium 199 with Hanks' salts and antibiotics), frozen at — 70°C, and then assayed on Vero cell monolayers to determine the amount of virus ingested. The remaining engorged mosquitoes were placed in 3.8-liter cardboard containers with netting on one end, and apple slices or a 7% sucrose solution were provided as a carbohydrate source. The containers were placed in plastic bags to maintain a high humidity and held in an incubator maintained at 26°C. Seven days after the infectious blood meal, approximately half of the mosquitoes remaining in each cage were sampled to determine infection and dissemination rates. Mosquitoes were cold anesthetized and triturated individually in 1 ml of diluent. For the first 10 mosquitoes sampled in each strain, the legs and bodies were triturated separately in 1 ml of diluent. The remaining mosquitoes were triturated intact. At 14 d after the infectious blood meal, the remaining mosquitoes in each cage were allowed to feed as a group on an anesthetized 3—5 wk-old ICR laboratory mouse (Charles River Laboratories, Wilmington, Mass.) The feeding status of each mosquito was recorded, then the specimens were triturated as described above for the day 7 samples. Mosquitoes were considered to be infected if virus was recovered from body tissues sampled at 7 or 14 d after the infectious blood meal. For mosquitoes that had their legs and bodies triturated separately, a mosquito that had virus recovered from its body (but not from its legs) was considered to have a nondisseminated infection limited to its midgut. In contrast, if virus was recovered from both body and leg suspensions, the mosquito was considered to have a
F4^ FQ-IO
F7 (Old laboratory strain) F5 F2 F5 F6-7 F2 F5 F3 F5 (Old laboratory strain) F2 F4 F7 F4
disseminated infection (Turell et al. 1984). We considered mosquitoes that were triturated intact and had a titer s l O 5 plaque-forming units to have a disseminated infection. Of the mosquitoes that had their legs and bodies assayed separately, 95% (164 of 173) with a body titer >10 5 plaque-forming units also had a disseminated infection, whereas 98% (589 of 602) of those with a titer 10 5 0 plaque-forming units in the whole-body suspension.
viremias of 1O 42 ^ 46 plaque-forming units/ml were consistently higher in the 10 strains of Ae. albopictus (range, 16—47%) than in the 7 strains of Ae. aegypti (range, 0—15%) (Table 3). Similarly, dissemination rates tended to be higher in the Ae. albopictus strains (range, 0—32%) than in the Ae. aegypti strains (range, 0—11%). For both species at both infectious doses tested, infection rates were similar at 7 or 14 d after the infectious blood meal (Tables 2 and 3). In contrast, dissem-
ination rates tended to be higher at 14 than at 7 d after the infectious blood meal (Tables 2 and 3). Because mosquitoes were allowed to feed in groups on the mice, it was not possible to calculate transmission rates. However, for virtually all of the strains (4 of 4 [100%] of Ae. aegypti and 7 of 8 [88%] of Ae. albopictus) in which an individual with a disseminated infection fed on a mouse, that mouse contained neutralizing antibodies to CHIK virus when bled 21 d later. Thus,
Table 3. Infection and dissemination rates in selected strains of Ae. albopictus and Ae. aegypti 7 or 14 d after ingestion of 10* 2 " 4 6 PFU/ml of blood of CHIK virus
•
No. tested Days after ingestion 7 14
Infection rate' Days after ingestion Total
Total 7
14
Ae. albopictui 47 48
GENTILLY OAHU MADAGASCAR SABAH SAO PAULO OKINAWA ZAMA POLK HOUSTON TAIWAN
85 70 25 35 71 25 50 30 80 20
86 81 26 36 58 25
171 151 51 129 50
30
35 33 34
32
32
64
114 60 176
28 30
28 13
14
25 9
ROCKEFELLER LAS VIRTUDES GENTILLY FOSTER DAKAR REX THAILAND
50 55 60 35 45 30 25
56 52 81 21 48 30 26
30 96 23
71
43 106 107 141 56 93 60 51
43 44 43
36
25 Ae. aegypti 18 16 5 6 2 3
13 12 11 0 6 0 0
0 2
47a
39ab 39abc 38abc 32abc 32abcd 28bcd 22bcde 20cde 16bcdef 15def 14def 9ef 4ef 4f 2f Of
Dissemination rate'J Days after ingestion Total 7
14
32 21
33
20 6 13 4
14 3 1 0
23 12
17 29 16 19
10
32a 22ab 16abcd llbcde 20abcd lObcde 17abcd 7bcde
9
6de
0
Ode
12
9
11 2 6 0 0
12 9 0 4 0 0
lObcde llbcde 6bcde
0
4de 2de Oe Ode
Rates followed by the same letter are not significantly different by a x test (« = 0.005). No adjustments were made for multiple comparisons. " Percentage of mosquitoes containing vins. b Percentage of mosquitoes with virus in their legs or, if legs not tested, that contained >10 5 0 plaque-forming units in the whole-body suspension.
52
JOURNAL OF MEDICAL ENTOMOLOGY
each strain that developed disseminated infections appeared to be able to transmit CHIK virus by bite. Discussion Strains of Ae. albopictus, regardless of their geographic origin, were more susceptible to infection with CHIK virus than were the strains of Ae. aegypti tested. Although nearly all strains of both species transmitted CHIK virus by bite, Ae. albopictus had relatively higher infection and dissemination rates, and appeared to be a more competent vector of CHIK virus than did Ae. aegypti. Because Ae. aegypti is considered to be the primary vector of CHIK virus in Asia and a secondary vector in Africa (Jupp & Mclntosh 1988), the introduction of a potentially more susceptible host, Ae. albopictus, into the Americas may increase the risk of epidemic transmission of CHIK virus in the Americas should this virus be introduced. The viremias to which mosquitoes were exposed in our study are comparable to those to which mosquitoes would be exposed in nature (Sarkar 1966, Jupp & Mclntosh 1988), and Ae. albopictus readily feeds on humans (Tempelis et al. 1970). In addition, Ae. albopictus has been implicated as a natural vector of dengue virus (Metselaar et al. 1980). This mosquito has extended its range in many areas, and should be considered a potential vector of CHIK virus. Tesh et al. (1976) also found variation in the susceptibility of geographic strains of Ae. albopictus to CHIK virus, with the OAHU strain most susceptible. Our results confirmed that this strain is highly susceptible to infection with CHIK virus, as it was the most susceptible and second most susceptible strain tested at viremias of 10 5 3 and 104-2"4-6 plaque-forming units/ml, respectively. However, the infection rates for the OAHU strain in our study were 97 and 39% at these viremias, respectively. These were considerably higher than the infection rates of only 44 and 8% at essentially identical doses of virus reported by Tesh et al. (1976). This difference in susceptibility probably was attributable to their use of a blood-virus suspension to expose mosquitoes rather than a viremic host (Jupp 1976, Meyer et al. 1983, Miller 1987, Turell 1988). Despite the significant differences between the two species and the large variation in infection rates within Ae. albopictus, no consistent pattern in infection or dissemination rates due to geographic origin of the strains was observed within either species. For example, although both the GENTILLY and the HOUSTON strains of Ae. albopictus were derived from the southern United States, they represent the most and nearly the least susceptible strains tested for this species. Likewise, although both the LAS VIRTUDES and REX strains of Ae. aegypti originated from specimens collected near San Juan,
Vol. 29, no. 1
Puerto Rico, they were among the most and least susceptible strains tested for this species. In addition, results were not always consistent within strains. For example, the POLK strain was one of the most susceptible when tested at 105 3 plaque-forming units/ml but was one of the least susceptible when tested at 104 2~4 6 plaque-forming units/ml. However, because of the numerous importations of these two species worldwide, the recent introduction^ ?) of Ae. albopictus into the Americas, and the relatively small number of mosquitoes often used to initiate a colony, it is not surprising that we did not find any consistent relationship between geographic origin of a strain and its susceptibility to infection with CHIK virus. We used a Southeast Asian strain of CHIK virus in this study. As reported by Tesh et al. (1976), Ae. albopictus strains were more susceptible to an Asian than to an African strain of GHIK virus. Thus, the use of a strain of CHIK virus of African origin may have produced different results. Additional studies, using Asian as well as African strains of CHIK virus and biochemical techniques such as isoenzyme patterns or DNA homology, may allow us to evaluate the relationship between vector competence and genetic relatedness among mosquito populations. Acknowledgment We thank G. B. Craig, Jr., W. Hawley, and L. Munstermann (University of Notre Dame); G. Clark, J. Freier, and C. Mitchell (Centers for Disease Control); and D. Strickman (U.S. Medical Component AFRIMS) for providing the various strains of Ae. aegypti and Ae. albopictus, without which this study could never have been accomplished. We also thank J. Kondig and his insectary staff for their assistance in rearing the mosquitoes, F. Malinoski for his assistance in infecting and handling the rhesus monkeys, and L. Durden and S. Gordon for their critical reading of the manuscript. In conducting the research described in this report, the investigators adhered to the Guide for the Care and Use of Laboratory Animals, as promulgated by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council. The facilities are fully accredited by the American Association for Accreditation of Laboratory Animal Care.
References Cited Banerjee, K., D. T. Mourya & A. S. Malunjkar. 1988. Susceptibility and transmissibility of different geographical strains of Aedes albopictus mosquitoes to chikungunya virus. Indian J. Med. Res. 87: 134138. Earley, E., P. H. Peralta & K. M. Johnson. 1967. A plaque neutralization method for arboviruses. Proc. Soc. Exp. Biol. Med. 125: 741-747. Gargan, T. P. II, C. L. Bailey, G. A. Higbee, A. Gad & S. El Said. 1983. The effect of laboratory colonization on the vector-pathogen interactions of Egyp-
January 1992
TURELL ET AL.: SUSCEPTIBILITY OF Aedes TO CHIK VIRUS
tian Culex pipiens and Rift Valley fever virus. Am. J. Trop. Med. Hyg. 32: 1154-1163. Hawley, W. A. 1988. The biology of Aedes albopictus. J. Am. Mosq. Control Assoc. 4: 1-39 (suppl.). Jupp, P. G. 1976. The susceptibility of four South African species of Culex to West Nile and Sindbis viruses by two different infecting methods. Mosq. News 36: 166-173. Jupp, P. G. & B. M. Mclntosh. 1988. Chikungunya virus disease, pp. 137-156. In T. P. Monath [ed.], The arboviruses: epidemiology and ecology, vol. 2. CRC, Boca Raton, Fla. Knudson, A. B. 1986. The significance of the introduction of Aedes albopictus into the southeastern United States with implications for the Caribbean and perspectives of the Pan American Health Organization. J. Am. Mosq. Control Assoc. 2: 420-423. Levitt, N. H., H. H. Ramsburg, S. E. Hasty, P. M. Repik, F. E. Cole, Jr., & H. W. Lupton. 1986. Development of an attenuated strain of chikungunya virus for use in vaccine production. Vaccine 4: 157-162. Mangiafico, J. A. 1971. Chikungunya virus infection and transmission in five species of mosquito. Am. J. Trop. Med. Hyg. 20: 642-645. Metselaar, D., C. R. Grainger, K. G. Oei, D. G. Reynolds, M. Pudney, C. J. Leake, R. M. Tukei, R. M. D'Offay & D.I.H. Simpson. 1980. An outbreak of type 2 dengue fever in the Seychelles, probably transmitted by Aedes albopictus (Skuse). Bull. W.H.O. 58: 937-943. Meyer, R. P., J. L. Hardy & S. B. Presser. 1983. Comparative vector competence of Culex tarsalis
53
and Culex quinquefasciatus from the Coachella, Imperial, and San Joaquin Valleys of California for St. Louis encephalitis virus. Am. J. Trop. Med. Hyg. 32: 305-311. Miller, B. R. 1987. Increased yellow fever virus infection and dissemination rates in Aedes aegypti mosquitoes orally exposed to freshly grown virus. Trans. R. Soc. Trop. Med. Hyg. 81: 1011-1012. Sarkar, J. K. 1966. Virological studies of haemorrhagic fever in Calcutta. Bull. W.H.O. 35: 59. Shroyer, D. A. 1986. Aedes albopictus and arboviruses: a concise review of the literature. J. Am. Mosq. Control Assoc. 4: 424-428. Tempelis, C. H., R. O. Hayes, A. D. Hess & W. C. Reeves. 1970. Blood-feeding habits of four species of mosquito found in Hawaii. Am. J. Trop. Med. Hyg. 19: 335-341. Tesh, R. B., D. J. Gubler & L. Rosen. 1976. Variation among geographic strains of Aedes albopictus in susceptibility to infection with chikungunya virus. Am. J. Trop. Med. Hyg. 25: 326-335. Turell, M. J. 1988. Reduced Rift Valley fever virus infection rates in mosquitoes associated with pledget feedings. Am. J. Trop. Med. Hyg. 39: 597602. Turell, M. J., T. P. Gargan II & C. L. Bailey. 1984. Replication and dissemination of Rift Valley fever virus in Culex pipiens. Am. J. Trop. Med. Hyg. 33: 176-181. Received for publication 1 April 1991; accepted 8 July 1991.
J. Med Entomol. 29(1): 49-53 (1992) ABSTRACT The relative susceptibility of selected strains of Aedes aegypti (L.) and Aedes albopictus (Skuse) fed on a viremic monkey to infection with chikungunya virus was determined. Infection rates were consistently higher in 10 strains of Ae. albopictus tested than in 7 strains of Ae. aegypti tested, regardless of the geographic location from which the strains originated or the dose of virus ingested. Similarly, virus dissemination rates were higher in the Ae. albopictus strains compared with the Ae. aegypti strains. For nearly all (11 of 12) strains tested of both species, groups of mosquitoes with one or more females with a disseminated infection transmitted virus by bite to weanling mice. Based on these studies, Ae. albopictus appears to be a more competent laboratory vector of chikungunya virus than does Ae. aegypti. KEY WORDS Insecta, Aedes spp., chikungunya virus, infection rates
Aedes aegypti (L.) is considered to be the principal vector of chikungunya (CHIK) virus in Asia, various studies indicate that this species has a high threshold of infection for CHIK virus (Jupp & Mclntosh 1988). Other studies have indicated that Aedes albopictus (Skuse) may be a potential secondary vector for this virus (Mangiafico 1971, Jupp & Mclntosh 1988). The recent introduction of this species into the Americas has raised concern that it may serve as a vector for exotic as well as endemic viruses (Knudson 1986). This species is a competent laboratory vector for numerous arboviruses (Shroyer 1986, Hawley 1988). In addition, Ae. albopictus can displace populations of Ae. aegypti in the southern United States (J. Freier, personal communication) and has demonstrated an ability to flourish in tree holes as well as in artificial containers (Hawley 1988). Because the potential for Ae. albopictus (Tesh et al. 1976) and Ae. aegypti (Banerjee et al. 1988) to transmit CHIK virus differs for strains isolated from different geographic locations, we evaluated 10 strains of Ae. albopictus and 7 strains of Ae. aegypti for their ability to transmit CHIK virus. Most previous studies on the ability of Aedes mosquitoes to transmit CHIK virus have used techniques such as droplet feeding, bloodsoaked pledgets, or membrane feeders to expose mosquitoes to virus. Virus infection rates in mosquitoes that are artificially exposed to virus may be significantly lower than in mosquitoes that ingested the same virus dose from a viremic animal (Jupp 1976, Meyer et al. 1983, Miller 1987, Turell 1988). Therefore, to use a more natural system and to ensure that mosquitoes were exALTHOUGH
posed to the same virus dose, we allowed up to 10 different strains of mosquitoes to feed concurrently on the same viremic rhesus monkey. Materials and Methods Mosquitoes. Ten geographic strains of Ae. albopictus and seven of Ae. aegypti were obtained from various sources (Table 1). With the exception of two strains that had been colonized for many years, attempts were made to use newly established colonies as much as possible. All strains were maintained at 26°C and high humidity according to procedures described by Gargan et al. (1983). Female mosquitoes were between 4 and 10 d old when used for infection trials. Virus and Virus Assay. Strain 15561 of CHIK virus was isolated from human serum in Thailand during the 1962 epidemic. It was passaged twice in primary green monkey kidney cells before its use in these studies (Levitt et al. 1986). Serial 10-fold dilutions of specimens were tested for infectious virus by plaque assay on Vero cell monolayers. Determination of Vector Susceptibility. Between 50 and 70 female mosquitoes from each strain were placed in 0.5-liter cardboard containers (one strain per container) with netting over one end. To ensure a similar virus exposure for each strain tested, mosquitoes in up to 10 of these containers were allowed to feed concurrently on each of three anesthetized rhesus monkeys that had been inoculated subcutaneously 24 or 48 h earlier with 0.2 ml of a suspension containing 107-108 plaque-forming units of CHIK virus. There were five feedings (two at 24 h and
JOURNAL OF MEDICAL ENTOMOLOGY
50
Vol. 29, no. 1
Table 1. Strains of Ae. aegypti and Ae. Albopictus Species
Strain
Collection location (yr collected)
Ae. albopictus
GENTILLY HOUSTON MADAGASCAR OAHU OKINAWA POLK SABAH SAO PAULO TAIWAN ZAMA LAS VIRTUDES REX ROCKEFELLER THAILAND GENTILLY FOSTER DAKAR
New Orleans, La. (1988) Houston, Tex. (1985) Antananarivo, Madagascar (1987) Honolulu, Hawaii (1971) Naha, Okinawa (1987) Polk, Fl. (1989) Sabah, Malaysia (1986) Sao Paulo, Sao Paulo, Brazil (1987) Kaohsiung, Taiwan (1989) Tokyo, Japan (1986) San Juan, Puerto Rico (1988) Puerto Rico (1987)
Ae. aegypti
Generation
Bangkok, Thailand (1989) New Orleans, La. (1988) Columbus, Ind. (1986) Dakar, Senegal (1988)
three at 48 h after inoculation with virus). Before each feeding attempt, blood was removed from the femoral vein to determine the viremia level. In addition, immediately after several of the feedings, six engorged mosquitoes were individually triturated in 1 ml of diluent (10% fetal bovine serum in Medium 199 with Hanks' salts and antibiotics), frozen at — 70°C, and then assayed on Vero cell monolayers to determine the amount of virus ingested. The remaining engorged mosquitoes were placed in 3.8-liter cardboard containers with netting on one end, and apple slices or a 7% sucrose solution were provided as a carbohydrate source. The containers were placed in plastic bags to maintain a high humidity and held in an incubator maintained at 26°C. Seven days after the infectious blood meal, approximately half of the mosquitoes remaining in each cage were sampled to determine infection and dissemination rates. Mosquitoes were cold anesthetized and triturated individually in 1 ml of diluent. For the first 10 mosquitoes sampled in each strain, the legs and bodies were triturated separately in 1 ml of diluent. The remaining mosquitoes were triturated intact. At 14 d after the infectious blood meal, the remaining mosquitoes in each cage were allowed to feed as a group on an anesthetized 3—5 wk-old ICR laboratory mouse (Charles River Laboratories, Wilmington, Mass.) The feeding status of each mosquito was recorded, then the specimens were triturated as described above for the day 7 samples. Mosquitoes were considered to be infected if virus was recovered from body tissues sampled at 7 or 14 d after the infectious blood meal. For mosquitoes that had their legs and bodies triturated separately, a mosquito that had virus recovered from its body (but not from its legs) was considered to have a nondisseminated infection limited to its midgut. In contrast, if virus was recovered from both body and leg suspensions, the mosquito was considered to have a
F4^ FQ-IO
F7 (Old laboratory strain) F5 F2 F5 F6-7 F2 F5 F3 F5 (Old laboratory strain) F2 F4 F7 F4
disseminated infection (Turell et al. 1984). We considered mosquitoes that were triturated intact and had a titer s l O 5 plaque-forming units to have a disseminated infection. Of the mosquitoes that had their legs and bodies assayed separately, 95% (164 of 173) with a body titer >10 5 plaque-forming units also had a disseminated infection, whereas 98% (589 of 602) of those with a titer 10 5 0 plaque-forming units in the whole-body suspension.
viremias of 1O 42 ^ 46 plaque-forming units/ml were consistently higher in the 10 strains of Ae. albopictus (range, 16—47%) than in the 7 strains of Ae. aegypti (range, 0—15%) (Table 3). Similarly, dissemination rates tended to be higher in the Ae. albopictus strains (range, 0—32%) than in the Ae. aegypti strains (range, 0—11%). For both species at both infectious doses tested, infection rates were similar at 7 or 14 d after the infectious blood meal (Tables 2 and 3). In contrast, dissem-
ination rates tended to be higher at 14 than at 7 d after the infectious blood meal (Tables 2 and 3). Because mosquitoes were allowed to feed in groups on the mice, it was not possible to calculate transmission rates. However, for virtually all of the strains (4 of 4 [100%] of Ae. aegypti and 7 of 8 [88%] of Ae. albopictus) in which an individual with a disseminated infection fed on a mouse, that mouse contained neutralizing antibodies to CHIK virus when bled 21 d later. Thus,
Table 3. Infection and dissemination rates in selected strains of Ae. albopictus and Ae. aegypti 7 or 14 d after ingestion of 10* 2 " 4 6 PFU/ml of blood of CHIK virus
•
No. tested Days after ingestion 7 14
Infection rate' Days after ingestion Total
Total 7
14
Ae. albopictui 47 48
GENTILLY OAHU MADAGASCAR SABAH SAO PAULO OKINAWA ZAMA POLK HOUSTON TAIWAN
85 70 25 35 71 25 50 30 80 20
86 81 26 36 58 25
171 151 51 129 50
30
35 33 34
32
32
64
114 60 176
28 30
28 13
14
25 9
ROCKEFELLER LAS VIRTUDES GENTILLY FOSTER DAKAR REX THAILAND
50 55 60 35 45 30 25
56 52 81 21 48 30 26
30 96 23
71
43 106 107 141 56 93 60 51
43 44 43
36
25 Ae. aegypti 18 16 5 6 2 3
13 12 11 0 6 0 0
0 2
47a
39ab 39abc 38abc 32abc 32abcd 28bcd 22bcde 20cde 16bcdef 15def 14def 9ef 4ef 4f 2f Of
Dissemination rate'J Days after ingestion Total 7
14
32 21
33
20 6 13 4
14 3 1 0
23 12
17 29 16 19
10
32a 22ab 16abcd llbcde 20abcd lObcde 17abcd 7bcde
9
6de
0
Ode
12
9
11 2 6 0 0
12 9 0 4 0 0
lObcde llbcde 6bcde
0
4de 2de Oe Ode
Rates followed by the same letter are not significantly different by a x test (« = 0.005). No adjustments were made for multiple comparisons. " Percentage of mosquitoes containing vins. b Percentage of mosquitoes with virus in their legs or, if legs not tested, that contained >10 5 0 plaque-forming units in the whole-body suspension.
52
JOURNAL OF MEDICAL ENTOMOLOGY
each strain that developed disseminated infections appeared to be able to transmit CHIK virus by bite. Discussion Strains of Ae. albopictus, regardless of their geographic origin, were more susceptible to infection with CHIK virus than were the strains of Ae. aegypti tested. Although nearly all strains of both species transmitted CHIK virus by bite, Ae. albopictus had relatively higher infection and dissemination rates, and appeared to be a more competent vector of CHIK virus than did Ae. aegypti. Because Ae. aegypti is considered to be the primary vector of CHIK virus in Asia and a secondary vector in Africa (Jupp & Mclntosh 1988), the introduction of a potentially more susceptible host, Ae. albopictus, into the Americas may increase the risk of epidemic transmission of CHIK virus in the Americas should this virus be introduced. The viremias to which mosquitoes were exposed in our study are comparable to those to which mosquitoes would be exposed in nature (Sarkar 1966, Jupp & Mclntosh 1988), and Ae. albopictus readily feeds on humans (Tempelis et al. 1970). In addition, Ae. albopictus has been implicated as a natural vector of dengue virus (Metselaar et al. 1980). This mosquito has extended its range in many areas, and should be considered a potential vector of CHIK virus. Tesh et al. (1976) also found variation in the susceptibility of geographic strains of Ae. albopictus to CHIK virus, with the OAHU strain most susceptible. Our results confirmed that this strain is highly susceptible to infection with CHIK virus, as it was the most susceptible and second most susceptible strain tested at viremias of 10 5 3 and 104-2"4-6 plaque-forming units/ml, respectively. However, the infection rates for the OAHU strain in our study were 97 and 39% at these viremias, respectively. These were considerably higher than the infection rates of only 44 and 8% at essentially identical doses of virus reported by Tesh et al. (1976). This difference in susceptibility probably was attributable to their use of a blood-virus suspension to expose mosquitoes rather than a viremic host (Jupp 1976, Meyer et al. 1983, Miller 1987, Turell 1988). Despite the significant differences between the two species and the large variation in infection rates within Ae. albopictus, no consistent pattern in infection or dissemination rates due to geographic origin of the strains was observed within either species. For example, although both the GENTILLY and the HOUSTON strains of Ae. albopictus were derived from the southern United States, they represent the most and nearly the least susceptible strains tested for this species. Likewise, although both the LAS VIRTUDES and REX strains of Ae. aegypti originated from specimens collected near San Juan,
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Puerto Rico, they were among the most and least susceptible strains tested for this species. In addition, results were not always consistent within strains. For example, the POLK strain was one of the most susceptible when tested at 105 3 plaque-forming units/ml but was one of the least susceptible when tested at 104 2~4 6 plaque-forming units/ml. However, because of the numerous importations of these two species worldwide, the recent introduction^ ?) of Ae. albopictus into the Americas, and the relatively small number of mosquitoes often used to initiate a colony, it is not surprising that we did not find any consistent relationship between geographic origin of a strain and its susceptibility to infection with CHIK virus. We used a Southeast Asian strain of CHIK virus in this study. As reported by Tesh et al. (1976), Ae. albopictus strains were more susceptible to an Asian than to an African strain of GHIK virus. Thus, the use of a strain of CHIK virus of African origin may have produced different results. Additional studies, using Asian as well as African strains of CHIK virus and biochemical techniques such as isoenzyme patterns or DNA homology, may allow us to evaluate the relationship between vector competence and genetic relatedness among mosquito populations. Acknowledgment We thank G. B. Craig, Jr., W. Hawley, and L. Munstermann (University of Notre Dame); G. Clark, J. Freier, and C. Mitchell (Centers for Disease Control); and D. Strickman (U.S. Medical Component AFRIMS) for providing the various strains of Ae. aegypti and Ae. albopictus, without which this study could never have been accomplished. We also thank J. Kondig and his insectary staff for their assistance in rearing the mosquitoes, F. Malinoski for his assistance in infecting and handling the rhesus monkeys, and L. Durden and S. Gordon for their critical reading of the manuscript. In conducting the research described in this report, the investigators adhered to the Guide for the Care and Use of Laboratory Animals, as promulgated by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council. The facilities are fully accredited by the American Association for Accreditation of Laboratory Animal Care.
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