Vector Competence of Aedes Texas, for Dengue

JouRNeloF THEAuprrclrv Mosqurro CoNrnor,Assocrerror.r VoL.3,No.3

VECTOR COMPETENCE OF AEDES ALBOPIC"US FROM HOUSTON. TEXAS, FOR DENGUE SEROTYPES1 TO 4, YELLOW FEVER AND ROSSRIVER VIRUSES

CARL J. MITCHELL,I BARRY R. INo DUANE J. GUBLER,

Diuision of Vector-Borne .Viral Diseases,-Centerfor Infectiotn Diseases,Centers for DiseaseControl, Publir Health Seruice,U.S. Department of Health and Human Seruices

ABSTRACT' A combination of virus infection and tra-nsmissionexperiments showed that a Houston, Texas.:llain.of Aedes albopictusis a competent vector for dengue (tiEN), yellow te"er iiF) ""a fio.*i River (RR) viruses. However, at 14 days incubation, DEN viruJinfection iaLs in a Puerto n1"* .trui" of Aedes aegypti we.resignificantly higher for each of the four DEN serotypes,except OnN-f , in"" i" Houston Ae' ahopictus fed_simultaneouslyon the same virus suspensions.ttre deg.ee oi.ott"f"Uo" between disseminatedDEN infection rates in Houston Ae. ahopictus and transmisiion to an in uitro system ranged from 42 to 88Vofor the four DEN serotypes.No lignificant difference was noted in YF virus infection rates or transmission rates in the two mosquito speciesfed on the samevirus suspensions and incubated for the sametime period. AIso, RR virus inlection and transmission rates in Houston and Hawaiian strains of Ae. albopictuswere generally comparable.

INTRODUCTION Only Fz and F3 Iaboratory generation females were used in our experiments. The Hawaii Ae. Since the discovery of Aedes albopictus albopictuscolony used for comparativepurposes (Skuse) in Harris County, Texas (Sprenger and in the RR virus experiments was from Makiki, Wuithiranyagool 1986), its presence has been Oahu, Hawaii, and in the Frg and Fzolaboratory documented in 12 states in the United States generations;its vector competencefor RR virus and in three statesin Brazil (Anonymous 1986a, had been determined previously (Mitchell and 1986b;Forattini 1986;CDC, unpublisheddata). Gubler 1987).The Ae. aegypti(Linn.) strain was Aside from its significance as pest a species,Ae. from the Rexville, Puerto Rico colony main- albopictus in the Western Hemisphere has tainedat the Centersfor DiseaseControl (CDC) arousedthe interest of public health authorities Iaboratory in San Juan. The generation history becauseof its known and potential vector rela- of the parent colony is unknown, but the strain tionship with several arboviruses of public had been colonized for about 4 years with peri- health importance(Shroyer 1986). Of chief con- odic additions of field-collected Ae. aegpti. A cern in the southernUnited States,and Central subcolony was established in our Fort Collins and South America is the impact that Ae. albo- insectary during March 1986,and F1,F2 and F3 pictus may have on the transmission and main- generations from this colony were used in the tenanceof dengue(DEN) and yellow fever (YF) experiments. viruses. We report here on experimental infec- Experimental procedure.All mosquitoeswere tion and transmission studies with these viruses rearedat 26.7 (+9.5"9), 80% RH, and a photo- and a strain of Ae. albopicfus from Houston, period of L:D 16:8. Three- to five-day-old fe- Texas. In addition, we have included Ross River maleswere used in the feedingtrials. Mosquitoes (RR) virus, an alphavirus that has recently ex- were allowed to feed on suspensionsconsisting its range into the Pacific and for tended basin of fresh DEN virus grown in Toxorhynchites which several geographicstrains of Ae. albopic- arnboinesis(Doleschall) or YF virus grown in fus have beenshown to be efficient experimental C6/36 cells or suckling-mousebrains, harvested vectors(Mitchell and Gubler 1987). on the day of feeding, diluted as appropriate, and mixed with equal volumes of washedhuman MATERIALS AND METHODS red blood cells. Procedures for preparing and feeding the YF virus suspensionwere described Viral and mosquito strains. The sourcesand in detail (Miller and Mitchell 1986);the follow- passage histories of the viral strains are shown ing procedure for preparing the DEN virus is in Table 1. The Houston Ae. albopictuscolony that used at the CDC, San Juan Laboratories. was established from 48 females collected as Recently emerged Tx. amboinesisfemales were adults and approximately 12 females and a few inoculated with seedvirus, incubated for 7 days males reared from larvae. all collected in Hous- at 26.7"C, and cold-anesthetized mosquitoes ton, Harris County, Texas, during March 1986. were triturated live in heat-inactivated calf serum at ratios of 0.1 to 0.2 ml per mosquito, 1P.O.Box 2087,Fort Collins,CO 80522. dependingon the number of mosquitosavailable 'GPO Box 4532.San Juan, PR 00936. and the volume of feeding suspensionrequired. 401 SEPTEMBEB1987 VEcroR ColapptpNcs oF AEDESALBoPIcrus

Table 1. Sourcesand passagehistories of viral strains. Virus Strain Source Location Year Passage DEN.1 1620 Human serum Puerto Rico 1985 Mosquito-1 DEN-2 1615 Human serum Puerto Rico 1985 Mosquito-1 DEN-3 r557 Human serum Mozambique 1985 Mosq.-l, C6l36-1 DEN-4 t632 Human serum Puerto Rico 1985 Mosquito-1 YF 788379 Hacmagogusspegazzini Trinidad 1978 c6/36-2 RR Human serum Rarotonga 1980 Mosquito-1

(Rosen Mosquito suspensionswere centrifuged at 4,000 with 0.17 rrl eachof tenfold dilutions and rpm for 30 min at 4"C, and the supernatantwas Gubler 1974)and calculatingthe mosquito in- removed,diluted as appropriate, and mixed with fectious doseso(MIDso)/ml (Reed and Muench equal volumes of washed red blood cells. Gen- 1938).Titrations of YF virus (Miller and Mitch- erally, suspensionscontaining freshly harvested ell 1986) and RR virus (Mitchell and Gubler DEN or YF viruses were fed undiluted and at 1987) were done by plaque assay in Vero cell 10- and 100-folddilutions. The feedingsuspen- culture. sions were warmed for 4 min at 37"C, and drops were placed directly on nylon netting covering RESULTS pint-sized cages containing the mosquitoes. Mosquitoes were exposedfor 15 min, and fully The susceptibility of Houston Ae. albopicttts engorgedspecimens were sorted,placed in cages, to per os infection with DEN viruses 1-4 is given \Vo sugar water, and incubated for appro- compared with that of Rexville Ae. aegypti in priate intervals at 26.7"C and80% RH. Mosqui- Table 2. Generally, DEN viral antigen was de- toes ingested RR virus by feeding on viremic tectable in head tissues by day 7 of incubation hamsters infected by subcutaneousinoculation in both species;however, Ae. albopictusdid not 48 to 96 hr prior to feeding (Mitchell and Gubler have detectable DEN-2 antigen at that time. 1987). Aedesaegyptl was not tested for DEN-4 antigen Yellow fever and RR virus transmission trials on day 7 becauseunexplained mortality in this were conducted by allowing mosquitoesto feed cohort reducedthe sample size, and we wished on 2-day-old suckling mice. Mice were moni- to keep the remainder for a longer incubation tored for 14 days for signs of illness or death. period. Dengue virus transmission was determined by DisseminatedDEN virus infection rateswere the in uitro feeding technique describedby Ait- compared in both speciesfor each DEN sero- ken (1977). Capillary tubes were loaded with type. There was no significant difference in in- approximately 5 pl of calf serum, and the pro- fection rates for any DEN serotypein Ae. aegypti boscis of a test mosquito was inserted in a tube and.Ae. albopictusincubated Iess than 14 days. following removal of the mosquito's wings. The However, at 14 days' incubation, the infection capillary tube was fixed in a Styrofoam@rack, rates inAe. aegyptiwere significantly greater (P and each mosquito was left in place for at least < 0.05,Fisher's exact test) for eachDEN sero- 30 min. The amount of feeding suspensionin- tlpe exceptDEN-I. gestedwas recorded, and the remainder of the Titers of DEN virus feeding suspensionsthat suspension was expressed onto a microscope would be expected to result in 50% infection slide, loaded into a calibrated capillary needle, rates in Ae. aegypti and Ae. albopictus that fed and injected parenterally into five, or occasion- on these suspensionswere calculated from the ally fewer, Rexville Ae. aegypti. These mosqui- data in Table 2 for DEN-I, DEN-2 and DEN-3, toes were given \Vo sugar water and incubated i.e., logle MIDoo/ml. DEN-4 was not included at26.7"Cfor 7 days.They were frozenat -70"C because of the paucity of data points. These until assayedfor virus. titers were virtually identical for DEN-I, 7.19 Mosquito infection with DEN and YF viruses and 7.20 in Ae. aegypti and Ae. albopictus,rc- was determined by examining head squashesfor spectively.However, the titer of the DEN-2 viral antigen by the direct fluorescent antibody feeding suspensionrequired to infect 50% of Ae. test (DFAT) (Kuberskiand Rosen1977). Some- aegyptiper os (6.57) was significantly lower (P times associatedcarcasses were sonicated and < 0.05, probit analysis) than for Ae. albopictw tested for YF virus by plaque assayin Vero cell (7.67). A significant difference (P < 0.05) also culture. AII assaysfor RR virus in mosquitoes was noted for DEN-3 virus, where the titers were done in Vero cell culture by methods re- required to infect 50% of Ae. aegypti and Ae. cently described(Mitchell and Gubler 1987). albopicttuwere 7.20and 9.16,respectively. Dengue stock virus and feeding suspensions We also tested four groups of Ae. albopictus were titrated by inoculating Tx. arnbionensis for their ability to transmit each of the four 462 JoURNALoF THE AuoRrcan Mosqurro CoNtnol AssocrATIoN Vor,.3, No. B

Table 2' Susceptibility of Rexville Aedes.oegyptiand.Houston A edesalbopittttsto infection per os

Infection ratesb Ae aegypti Titef Ae. albopictus Dengue Days incu- feeding No. % virus No. bation suspension tested pos. tested I 9.0 20 45 20 60 9.2 0 19 t4 58 6.6 60 38 60 23 7.6 58 67 60 70 8.9 0 25 r00 9.0 57 /D 68 85 9.2 2l ,1 100 40 98 7.6 20 25 0 7.6 0 20 0 13 6.3 31 10 60 T2 7.4 11 36 54 26 9.0 5 40 37 23 t4 5.6 33 24 34 3 6.6 T4 I 35 I7 7.6 27 74 32 53 7.6 20 6D 38 45 8.4 25 92 23 74 7 8.3 20 10 0 8.4 0 20 15 t4 6.3 32 4L 60 27 7.4 60 68" 59 22 8.1 25 u 15 53 8.3 60 47" 53 38 1 8.4 IL 34 4T 7 8.0 0 20 0 9.2 0 20 5 13 6.2 13 0 60 I

7.4 14 t4 60 Id 8.0 11 .{5 59 39 t4 7.9 25 76 19 43 9.2 6 50 42 29 " lo916MIDss/ml. b Based on detection of DEN viral antigen in head tissues " by DFAT. Records for the numbers fed and tested from these groups suggestthat a labelling error occurred; therefore, these data probably should be reversed.

DEN viruses following exposureto infection per Table 3. Day-14 infection and transmission rates in os and 14 days of incubation. Correlation be- the Houston strain of Aedesahopi.ctus orally exposed tween disseminatedinfection rates, i.e., mosqui- t" tn" i"". r"t"typ".. positive toes with head squashes, and virus Den- transmission (Table rates rangedfrom 42to 88% gue Meal titer No. No. trans- 3). sero- logro infectedY mittingl/ Yellow fever virus infection and transmission type MID5a/ml no. tested no. infected rates in Ae. aegypti and. Ae. ahopirtu.s are com- 1 8.9 25/25(t$O%l 2L/24(e8%) pared in Table 4. Both specieshad comparable 2 8.4 23/25(ezVo) r7/23(74%') infection (70 and 80%) and. transmission (4G 3 8.1 16/25(6470) 8/r5 (53%) and 55%) rates 11 days after feeding on a meal 4 7.9 L9/25(76%) 8/L9(42Vo\ containing 106?MIDoo/ml " of YF virus. No sig- Based on detection of dengue viral antigen in nificant differences (P > 0.05. Fisher's exact mosquito head tissues by DFAT. b test) were noted in the infection rates or trans- Determined by the in uitro feeding technique of mission rates of the two speciesfollowing inges- Aitken (1977). tion of similar amounts of virus and incubation for the same time period. and Ae. albopictus fed simultaneously on a meal In addition to the data on YF summarized in containing 104'eMIDso/ml of YF virus. On day Table 4, data were obtained on infection thresh- 11of incubation,4 of 25 (L6%) Ae. acgyptibodres olds and virus dissemination rates in Ae. apgypti contained virus as compared to I of 25 (4Vo) Ae. SEPTEMBER 1987 VEcroR CoupprnNcn or Asnss ALBoPIcrus 463

Table 4. Infection and transmission rates in Aedesaegypti andAedcs albopictusfed simultaneously on yellow fever virus suspensions. No. infected"/no. tested No. transmittin* / no. infected Meal titer Extrinsic logroPFU/ml incubation Ae. aegypti Ae. ahopictus Ae. acgypti Ae. ahopirttts

b.7 11 days 20/25(80%) 2r/30 (70%) 6/13(46%) 6hr (55%) 6.7 14 days L/L (t00Vo) 3a/46(74Vo) 0/L 4/28(L4%) 5.9 11 days t0/24(42Vo) 19/30(63%) 3/8 (38%) r/r4 (1%) 5.9 14 days 3/3 (roo%) L3/20(65%) L/3 (33%) r/8 (r3%) 5.0 14 days L2/30(40%) 6/20 (30%) 3/7 (43%) 0/6 'Based on detection of yellow fever viral antigen in mosquito head tissuesby DFAT' b Yellow fever virus transmission was assayedby allowing mosquitoesto feed on 2-day-old mice. ahopirtus. Yellow fever viral antigen was de- toes can transmit these viruses by 14 days post- tected in I of.25 Ae. oegypti heads and none of infection (Tables 2 and 3)' Some Ae' albopictus 25 Ae. albopirtlusheads. On day 14 of incubation, had disseminatedinfections of DEN-I, DEN-3 4 of 10 (407o)Ae. aagwti and 8 of 55 (L\Vo)Ae. and DEN-4 by day 7 postinfection,thus.sug- albopictusbodies contained YF virus, and 1 of gesting that a portion of the mosquitoesmay be L0 GO%)and 1 of 55 (2%) headswere positive able to transmit virus at this time. The Houston for antigen. The observeddifferences in infec- strain of Ae. albopictus was significantly less tion and dissemination rates between the two susceptible to per os infection with DEN-2, speciesare not significant (P > 0.5). All mos- DEN-3 and DEN-4 virusesthan wasAe. aegypti, quitoes were given an opportunity to refeed on but susceptibilities to DEN-1 virus were com- suckling mice to test for virus transmission; parable. The relative susceptibility of the two however, since infection rates were low, there speciescan be expected to vary depending on were few mosquitoes with disseminated infec- the origin ofthe geographicstrains (Gubler and tions amongthose that refed. The single infected Rosen1976, Gubler et al. 1979). Ae. aegypti that refed on day 11 successfully The DEN viruses also varied in their infectiv- transmitted virus; none of the remaining Ae. ity. In general,DEN-I and DEN-2 viruses were ocgypti and Ae. ahopicttts that refed had dissem- most infectious, DEN-3 intermediate, and inated infections. On day 14, the single Ae. DEN-4 the least infectious. Such variation in aegpti with a disseminated infection did not the infectivity of DEN viruseshas beenobserved refeed;the single Ae. albopictu.swith a dissemi- previously (Gubler and Rosen 1976).Transmis- nated infection refed but did not transmit virus. sion rates of the four DEN viruses also were Ross River virus infection and transmission different (Table 3); however,this variation may rates in Houston and Hawaii Ae. ahopictus arc be due to either viral strain variation or the titer compared in Tables 5 and 6, respectively.Both of the infective meal. Experiments were not strains were susceptibleto infection per os, al- done to answer this question. though the Hawaii strain was significantly (P < The DEN virus infection rates reported here 0.05) more susceptiblethan the Houston strain are generallyhigher than those found by Gubler in one feeding trial in which the hamster was and Rosen (1976). The titers of our feeding circulating 1062Vero cell plaque-forming units suspensionswere sometimes higher, and the use (PFU)/ml in the blood at the time of feeding. of fresh virus suspensionsthat had not been Both speciescould transmit virus on day 7 of frozen (Miller and Gubler, unpublished data) incubation, but transmission rates were higher probably accounts for the observeddifferences. on day 14 (Table 6). In two instances,RR virus Severalinvestigators have shown that infection transmission rates were significantly higher (P rates for a variety of arboviruses generally are < 0.05)among Hawaii Ae. albopictr,rcthan among Iower in mosquitoesfed virus suspensionswhen the Houston strain. compared with mosquitoes fed directly on vi- remic hosts with comparable titers (Mitchell presentedhere concern- DISCUSSION 1983).Therefore, data ing thresholds of infection for DEN and YF Geographic strains of Ae. albopictus arc viruses cannot be extrapolated and applied di- known to vary in their ability to becomeinfected rectly to field situations. It follows that we can- with dengue viruses by the oral route (Gubler not draw any firm conclusionsabout the suscep- and Rosen 1976). Our results show that the tibility thresholds of Ae. albopicfusto infection Houston strain of Ae. albopictus is susceptible with DEN and YF viruses in relation to virus to infection by eachDEN virus by the oral route titers that might be encountered when feeding and that a high proportion of infected mosqui- on viremic humans.Nonetheless, the compari- 464 JounNeL oF THE AMuRrclr{ Mosqurro Conrnor, AssocrATroN VoL.3,No.3

Table 5. Ross River virus infection rates in Hawaiian and Houston strains of Aedes ahopirtus. Day-7 incubation Day-14incubation Titef of Hawaii Houston infective Hawaii Houston meal n (Vo infect.\ n (% infect.) n (% infect.) n (% infect.) 4.5 _b 24 (33) 39(13) 5.2 50(90)" 50 (70) 50 (90)" 50 (70) 5.8 20(100) 20(65) 12(58) 14(79) 7.r 20 (100) 20 (1oo) 17(100) 24 (96) 1.O 50(e8) 50 (100) 46 (e8) 50(e8) " LogreVero cell PFU/ml. b Not done. " Differs significantly from other strain tested the sameday; P = 0.05 in Fisher's exact test.

Table 6. Ross River virus transmission rates by Hawaiian and Houston strains of ,Aedesalbonictus. Day-7 transmission Day-14 transmission Titer" of Hawaii Houston Hawaii infective Houston meal nb (% trans.) n (% trans.) n (% trans.) n (% trans.) 5.2 23 (43) 27 (33) 35 (77)" 25 (52) 5.8 12(75) 6 (50) 5 (100) 6 (67) r-l 10(e0) 17 (53) e (100) 11(64) 7.6 27(67)" 40 (38) 31(94) 36 (78) " Logls Vero cell PFU/ml. o Number of infected mosquitoesthat refed. " Differs significantly from other strain test€d the sameday; P < 0.0b in Fisher's exact test. sonsamong speciesof mosquitoesand strains of 4 days Iater and that maximum transmission virus are quite valid since any reduction in sen- efficiency was reachedby 10 to 13 days postin- sitivity attributable to the artificial feedingtech- fection. Our results suggest that the Hawaii nique should be the samein the paired compar- strain ofAe, albopictusmay sometimesbe a more isons. efficient experimental vector of RR virus than Our results show that Houston Ae. ahopicttn is the Houston strain. Mitchell and Gubler and Rexville A e. aegypti arereadily infected with (1987) previously showed that geographic YF virus by the oral route and that virus trans- strains of Ae. albopicfusmay vary in their vector mission rates are similar and substantial (55 and competencefor RR virus. 46To,tespectively)on day 11postinfection. Also, In view of the known and potential vector the strains of the two speciestested have similar relationship of.Ae. albopictuswith several arbo- thresholdsofinfection. Dinger et al. (1929)pre- viruses,its establishment in the Western Hemi- viously demonstrated that Ae. albopictus from sphere is a justifiable cause for concern. Java could transmit YF virus; however,since Whether its presence in the United States mosquitoes were tested in groups, it was not increasesthe risk of epidemic denguetransmis- possible to quantify infection and transmission sion is a point that may be debated since Ae. rates. oegypti already is present in many of the same The Houston strain of Ae. albopictus is also areas.However, the fact that all four DEN vi- an efficient experimental vector of RR virus. ruses can be transmitted transovarially by Ae. Infection rates approached 100% following the albopictus under experimental conditions (Ro- ingestionof high-titeredblood meals,and virus sen et al. 1983) wanants concern about its po- transmissionrates also were high (52 to 78Vo) tential as a reservoir for endemic dengue. by day 14 postinfection.Both the Hawaii and The situation in Central and South America Houston strains of Ae. albopiclfuswere also ca- and the islands of the Caribbean appears more pable of transmitting RR virus by day 7 postin- ominous. Aed.esalbopictus may contribute to fection. These results are in general agteement dengue transmission and maintenance, and in with those concerning RR virus in Ae. uigilax certain areas, has the potential of bridging the (Skuse),a primary vector in Australia and per- gap between jungle and urban yellow fever haps Fiji. Kay (1982) showed that Ae. uigilax cycles.The speciesmay becomeabundant in the infected per os could transmit RR virus by bite forest fringe and adjacent urban areas.Whether 465 SEPTEMBER 198? VEcroR CoupptnNcp os Agoss ALBoPICTUS it might also become established in jungle foci 28:1045-1052. J. and L. Rosen. 1976. Variation among Hietnagogus-transmittedYF remains to be Gubler, D. of geographicstrains of Aedes albopicttzsin suscepti- seen. Ultity to infection with denguevirus. Am. J. Trop' Med. Hyg. 25:318-325. tgAZ. Three modesof transmission of Ross ACKNOWLEDGMENTS Kay, B. H. Riuer uirus by Aedes uigilor (Skuse). Aust. J. Exp. We are grateful to Dr. Donald Eliason and Biol. Med. Sci. 60:339-344. A simple tech- Mr. MoisesMontoya, CDC, Ft. Collins, for pro' Kuberski, T. T. and L. Rosen. L977' of dengue antigen in mos-- Fr Iaboratory generation eggsofHouston nique for the detection viding quitoes by immunofluorescence.Am. J. Trop. Med' from which we established our Ae. albopictus, Hyg. 26:533-537. colony. Dr. Donald A. Shroyer, Florida Medica- Miller, B. R. and C. J. Mitchell. 1986' Passageof Entomology Laboratory, Vero Beach, kindly yellow fever virus: Its effect on infection and trans- provided eggsof Hawaii Ae. ahopirtus when he mission rates in Aedcs aegypti mosquitoes' Am. J. visited our laboratory en route from Hawaii, and Trop. Med. Hyg. 35:1302-1309. he also sent a preprint of his useful manuscript Mitchell, C. J. 1983.Mosquito vector competenceand to us.Mr. Ray Bailey,CDC, Ft. Collins,provided arboviruses,p. 63-92.ln: K. F. Harris (Ed'), Current his usual cheerful and efficient analysesof data topics in vector research. Praeger Scientific, New for statistical significance,and Ms. Carol Frank, York. C. J. and D. J. Gubler. 1987.Vector compe- of the same laboratory, typed the manuscript. Mitchell, tence of geographicstrains ofAedes albopittw and' Aedespolynesiensis and certain other Aedes (Stego' virus' J. Am. Mosq, REFERENCES CITED rnyio) mosquitoesfor RossRiver Control Assoc.3:142-147. Aitken,T. H. G. 1977.An in uitro feedingtechnique Reed, L. J., and H. Muench. 1938.A simple method for artificiallydemonstrating virus transmission by of estimating fifty percent endpoints' Am' J. Hyg. mosquitoes.Mosq. News 37:130-133. 27:493-497. Anonymous.L986a. Aedes albopictus infestation- Rosen,L. and D. Gubler. 1974.The use of mosquitoes UnitedStates, Brazil. MMWR 35:493-495. to detect and propagatedengue viruses. Am. J. Trop. Anonymous.1986b. Update: Aedes abopirtus infesta- Med. Hyg. 23:1153-1160. tion-United States.MMWR 35:649-651. Rosen,L., D. A. Shroyer, R. B. Tesh, J. E. Freier ani Dinger,J. E., W. A. P. Schuffner,E. P. Snijdersand J. C. Lien. 1983.Transovarial transmission ofden- N. H. Swellengrebel.1929. Onderzoek over Gele gue viruses by mosquitoes: Aedes ahopictw and' Koorts in Nederland(Dedre Medeeling). Nederl. Aedes ozgypti. Am. J. Trop. Med. Hyg. 32:1108- Tijdschr.V. Geneesk.73:5982-5991. 1119. Forattini, O. P. 1986.Identificacio de Aedes(Stego- Shroyer, D. A. 1986..Aedesalbopi.ctus and arboviruses: rnyin) ahopi.ctus(Skuse) no Brasil. Rev. Saude. A concise review of the literature. J. Am. Mosq. Pnbl.20:244-245. Control Assoc.2:424- 428. Gubler,D. J., S. Nalim, R. Tan, H. Saipanand J. Sprenger,D. and T. Wuithiranyagool. 1986.The dis- SuliantiSaroso. 1979. Variation in susceptibilityto coveryand distribution of Aedesolbopictus in Harris oralinfection with dengueviruses among geographic County, Texas. J. Am. Mosq. Control Assoc. 2: strainsof Aed.esaegypti. Am. J. Trop. Med. Hyg. 2t7-2t9.