JoURNALoF THE Aunnrcln Mosqutro Cottnor, AssocllTron Vol. 3, No. 2

VECTOR COMPETENCEOF GEOGRAPHICSTRAINS OF ALBOPICTUS AND AEDES POLYNES/ENS/S AND CERTAIN OTHER AEDES (STEGOMYIA)MOSQUTTOES FOR ROSS RrVER VrRUS

CARL J. MITCHELL' exo DUANE J. GUBLER,

Diuision of Vector-BorneViral Diseases,Center for Infectious Diseases,Centers for DiseaseControl, Public Health Seruici, U.S. Department of Health and Human Seruices

ABSTRACT. The vector competenceof geographicstrains of Aedesalbopictus and. Ae. polynesinnsisand, strains of Ae. pseudoscutellarisand Ae. aegypti was assessedfor Ross River (RR) virus, the etiologic agent of epidemicpolyarthritis. Strains of Ae. polynesiensrsfrom Fiji, Rarotonga,Samoa and Tahiti were the most susceptible to infection per os (MID66 < 1012 Vero cell plaque-forming units [PFU]/blood meal). Virus transmission data were variable, but all strains except the one from Fiji transmitted virus at 14 to 2I days postinfection. Shanghai and Hawaii Ae. ahopictus and Fiji Ae. pseudoscutellnriswere also highly susceptibleto per os infection with RR virus (MID561020 h 1026PFU). Singapore and Sri Lanka Ae. albopictusand Fiji Ae. aegypti were the least susceptible(MIDuo 1040to 1042PFU). With one exception, virus transmission rates for Ae. pseu.doscutellarisand Ae. aegypti and the four strains ofAe. albopictusranged from 52 to 700%. A total of 4,718 third- and fourth-instar larvae from the second gonotrophic cycle of potentially infected femaleswere tested for RR virus in 39 pools. Infection rates in parental femalesranged from 87 to 100%in Ae. albopictus,Ae. pseu.d,oscutellarisand Ae. polynesiensisand 40 to 48% in Ae. oegypti.Virus was not isolated from larval progeny.

INTRODUCTION tor of RR virus in Fiji and New Caledonia(Aus- tin et al. 1979,Marshall and Miles 1984),but its (RR) is an alphavirus and distribution in the Pacific doesnot extend east- polyarthritis the etiologic agent of epidemic ward beyondthe Fiji Islands.Aedes polyn'esiensis (EPA). geographic Until 1979,the known distri- Marks was the probable vector of RR virus in bution of RR virus was limited to Australia, the (Rosen et al. 1981, Gubler New Guinea, New Britain and the Solomon 1981),and it is the only mosquitospecies from Islands, all in the western half of the Pacific which virus was isolatedduring the 1979-1980 (Tesh basin et al. 1975).In April 1979,cases of outbreaksin the Pacific. Gubler (1981)showed EPA were reported from an area close to Nadi that a strain of Ae. polynesiensisfrom Raro- International Airport, Viti Levu, Fiji. The epi- tonga, Cook Islands, is an efficient vector of RR demic spread throughout the island group, re- virus under experimental conditions. This new sulting in an estimated 50,000 clinical cases virus-vector associationand epidemiologicdata (Aaskov et al. 1981). Several thousand addi- suggestinga man-mosquitocycle for RR virus tional cases resulted from further spread to in the outbreaks in American Samoaand Raro- AmericanSamoa in August 1979,to Futuna and tonga (Tesh et al. 1981,Rosen et al. 1981),are Wallis Islands in November 1979,and to New indicative of the potential for further spread of Caledonia,Tonga, and the Cook Islandsin Jan- the virus. (Marshall uary-February1980 and Miles 1984). We report here on the vector competenceof Although RR virus has been isolated from several geographic strains of Ae. albopictus several speciesof wild-caught mosquitoesin (Skuse) and Ae. polynesiensisand one strain Australia and severalspecies have been shown eachof Ae.pseudoscutellaris (Theobald) andAe. to be capableof transmitting the virus experi- aegypti(Linn.) from Fiji. Each of these species (Skuse) mentally, Aedes uigilax and Culex an- feeds readily on humans, has the potential for nulirostrisSkuse have been considered to be the rapid populationincreases and, with the excep- (Kay principal vectors et al. 1982).The distri- tion of Ae. albopictus,is presentin areaswhere bution of the latter speciescoincides with en- outbreaksof EPA have occurred.Among these zootic-endemicRR virus activity in Australia species,Ae. aegypti and Ae. polynesiensisprevi' and New Guinea,and it occurson all the Pacific ously have been shown to be capableof trans- island groups where caseshave been reported. mitting RR virus under experimental conditions (1984) However, Marshall and Miles suggest (Kay et al. 1979,1982; Gubler 1981). that Cr. annulirostris may be more important in maintaining primary cyclesthan as an epidemic vector. Aedesuigilax probably was the main vec- MATERIALS AND METHODS Virtts strain. We used a Rarotonga strain ol It was I P.O. Box 2087,Fort Collins,CO 80522. RR virus obtained from Dr. Leon Rosen- ' G.P.O.Box 4532,San Juan, PR 00936. isolated from human serum by inoculation into JUNE 1987 Vncron Coupntptcr ot Asoss 143

Table 1. speciesand strains used in Ross River virus infection and transmission experiments. Species Geographicorigin Laboratory colony generation Aedesahopictus Makiki, Oahu,Hawaii Fz,Fro, Frz Colombo,Sri Lanka Fz Singapore F2 Shanghai,China Beijing insectary colonv, F12s Shanghai,China F2 Ae. polynesiensis Fiji colonizedsince 1981" Rarotonga colonizedsince 1980" Samoa colonizedsince 1978' Tahiti colonizedsince 1969' Ae. pseudoscutellaris Fiji colonizedsince 1981' Ae. aegypti Fiji colonizedsince 1981' ' Generationhistory unknown.

Toxorhynchites atnboinensis (Doleschall) in fectedmice typically displayedhind leg paralysis March 1980.Our virus seedpools were prepared betweendays 6 and 9 postinfection. in heat-inactivatedfetal calf serum (FCS) fol- In one experiment,progeny of six lots of in- lowing one further passagein Tx. amboinensis. fected mosquitoes were tested to determine Mosquitaes.Four strains of Ae. polynesiensis, whethertransovarial transmission (TOT) of RR one strain eachof Ae. pseu.doscutellarlsand Ae. virus had occurred.Following an infective feed, aegypti, and five strains of Ae. albopictu.swere mosquitoes were allowed to oviposit and were used. Their origins and colonization histories then refed on uninfected hamsters on day 14 of are shown in Table 1. Mosquitoes were reared incubation. Eggs from the second oviposition in environmentalchambers at 26 + 1'C. Humid- cyclewere collected17 to 20 days following the ity in the chamberswas maintained,at 80 + \Vo infective meal, conditioned in the usual manner, RH, and the light:dark cycle was set at 16L:8D. hatched, and the resultant larvae were reared in Experimental procedure.Viremia curves were an incubator at 20"C and 15L:9D. determined in 6- to 10-week-oldGolden Svrian Adult mosquitoeswere disrupted by sonic en- hamsters inoculated subcutaneouslywith ap- ergy in 1 ml of BA-1 diluent (0.2 M Tris, pH proximately 10nVero cell plaque-forming units 8.0, 0.15 M NaCl, 1% BSA, 10 mglliter phenol (PFU) of RR virus. Ten hamsters were inocu- red, 50 pg/ml Gentamicin,' and 1 pg/ml Fungi- lated with virus, and two were sacrificed each zone). Suspensionswere centrifuged at 2,000 day and bled by cardiac puncture for viremia rpm for 20 min. The supernatant was frozen at determinations. Subsequently, 3- to 5-day-olc -70'C until tested.Third and fourth instar lar- mosquitoeswere allowed to feed for t hr or Iess vae were rinsed twice in M199 + 20VoFCS and on viremic hamstersthat had been anesthetized ground in pools of 125 or less in 2 ml of the with 0.15 cc of Ketamine HCI (100 mglml). same diluent, then centrifuged for 20 min at Hamsters were bled by heart puncture at the 2,000 rpm. Aliquots were frozen and subse- completion of feeding, and sera were collected quently testedin Vero cell culture. Blood sam- and frozen at -70"C until titrated. Engorged ples from hamsterswere taken by heart punc- mosquitoes were sorted and placed in gallon ture, allowed to clot, and centrifugedat 1,500 cages,provided with oviposition dishes contain- rpm for 15 min. Serawere dispensedinto screw- ing strips of moist paper, and incubated at 26J cap vials and storedat -70"C. + 0.5"C and.80VoRH. Some groups of mosqui- Specimens were screened for virus or were toes were fed simultaneously on the same vi- titrated as appropriate. Viral assayswere done remic hamster. A single anesthetized hamster by inoculating Vero cell cultures and counting wasplaced on top ofthree Plexiglasocages (1b.2 plaques.Briefly, tenfold dilutions were made in x 7.6 x 2.5 cm) covered with netting and con- BA-1, and samples(0.1 ml) wereinoculated into taining the mosquitoes.A few mosquitoeswere Vero cell cultures in six-well plates, adsorbed infected by feeding on suckling mice that had for t hr at 37'C, and overlaid with 1% Noble been inoculated subcutaneouslywith approxi- agar in M-199 supplementedwith 2% FCS, 2.0 mately 105to 106TCID56 24 hr earlier. g/liter of NaHCO3,150 pglml of DEAE-dextran, To assayfor virus transmission,we allowed mosquitoesto refeedindividually on l- to B-day- old suckling mice following appropriate periods 3 Use of trade names or commercial sources of incubation, is for The mosquitoeswere frozen, and identification only and does not constitute endorse- mice that were bitten were marked and returned ment by the Public Health Service or by the U.S. to their mothers and observedfor 14 davs. In- Department of Health and Human Services. r44 JouRNer.oF THE AupnrclN Moseurto CoNtnoL AssocratroN Vor,. 3,No. 2 and 1:4,000neutral red. Cell cultureswere then The results of experiments concerning the examined for 10 days for characteristic plaques. susceptibility of mosquito speciesand strains to Blood samplesfrom viremic suckling mice used per os infection with RR virus are summarized to infect mosquitoeswere assayedin the C6/36 in Tables 2 and 3. Since infection rates are static clone of Ae. albopictu.scells using methods pre- and since infected individuals are readily de- viously described (Rosen et al. 1981, Gubler tectable in our assay system by day 7 postfeed- 1981).Tests were not done to determine the ing, no distinction is made betweengroups with relationship betweenTCIDso and Vero cell PFU different incubation periods when analyzing the for RR virus; however, this information would infection rate data. Where possible, MIDso val- not alter the conclusionsdrawn. ueswere calculatedby probit regressionfor each The virus infection rate in mosquitoes, ex- speciesand strain fed on viremic hamsters.The pressedas a percentage,is the proportion of Iimited data for Ae. polynesiensisfed on suckling mosquitoestested that containedvirus. The IDso mice(Table 2) areexcludedbecause mouse blood valuewas estimated using probit regression.The sampleswere titrated in C6/36 cells, and titers virus transmissionrate, alsoexpressed as a per- wereexpressed as TCIDso.The MID59values are centage,is the proportion of infected mosquitoes based on the assumption that each mosquito that transmitted virus upon refeeding after a ingested5 pl ofblood. suitableextrinsic incubationperiod. Differences The MIDso valuescould not be determined for in infection rates between speciesfed simulta- strains of Ae. polynesiensisbecause 83% or more neouslyon the sameviremic hamster were tested becameinfected after feeding on the lowest ti- for significance by Fisher's Exact Test (Snede- tered blood mealstaken (Table 2). However,it cor and Cochran1967). is obviousthat these values would be below 1012 Vero cell PFU for eachof the four strains tested. RESULTS MIDso values (and 95% confidence limits) for Golden Syrian hamsters,6 to 10 weeks old, the other speciesand strains follow: Ae. pseu- were viremic on days 1 through 4 following doscutellaris1020 (101'6, L02'a); Ae. aegypti l0a2 subcutaneousinoculation of RR virus. Titers in (103e,104'5); Beijing Ae. albopirtus 102'5(10r'7, Vero cell PFU/ml were 1027and 103'5on day-l, 103'), Hawaii 102'6(10r'7, 103''), Shanghai 102'a 1060and 106'1on day-2,1066 and 1073on day-3, (100e,103'6), Singapore 104'0 (103'3, 10a'8), and Sri and 105.5and 106'6on day-4 postinoculation. Lanka 104't(103'1, 105'5). Paired comparisons be- Virus was not detectedon dav-5. tween MIDso values indicate that those of Beij-

Table 2. Ross River virus infection rates in geographicstrains ofAedes polyncsicnsisand one strain each of Ae. pseudoscutellaris and.Ae. aegypti fed.on viremic hamsters or suckling mice. Ae. oseudo- Ae' MEYPtt Ae. polynesicnsis r"it"llori^ Fiji Fiji Fiji Rarotonga Samoa Tahiti Titer of infec- tive meal' n (Voinfect.\ n (% infect.) n (% infect.) n (% infect.) n (% infect.) n (% infect.) 2.4 19b (5) 3.4 8o (88) 3.5 18b(83) 4.0 43b (37) 4.5 16b(93) 7b(1oo) 4.6 5b(100) 4.7 21b(1oo) 5.5 2b (1oo) 5.7 7" (100) 15 (93) 6.0 48 (40) 6 (100) 6.2 6.6 48 (48) 8 (100) 6.8 18 (100) 6.9 6 (100) I (100) 7.0 71 (9e) 1.J 5 (100) 23 (100) 8.3 8.5 30 (83) e (100) 8 (100) 5 (100) 'Titers from2.4 through ?.3 are logroVero cell PFU/ml from viremic hamsters;titers 8.3 and 8.5 are TCIDg/ ml from viremic sucklingmice. b Infection rates determined on day 7 of incubation; all others determined on days 14 to 2l of incubation. JUNE 1987 Vncton CoupnrnNcn ot Aooss 145

Table 3. Ross River virus infection rates in geographicstrains of Aedesahopictus fed on viremic hamsters and incubated 13 to 21 days.

Shanghai Frzo Shanghai F, Hawaii Singapore Sri Lanka Titer of infec- tive meal" n (% infect.\ n (% infect.) n (% infect.) n (Voinfect.) n (% infect.)

D.D 20(5) 4.0 53 (23) t. I 56 (16) 4.9 rs (74) 32 (53) 15(87)b b.u 40 (65) 50(4) 5.2 41 (85) 35 (37) 20(o) 5.3 30(7) o.o 25(t2) 5.6 37 (100) a.r 11 (82) 18 (89) 6 (100)b 6.0 39 (87) 6.2 59 (53) 6.3 8 (100) 14(100) I (89)b 6.5 60 (100) 5r (47) 6.7 20(80) 6.9 24 (96) 7.6 40 (95) 7.7 50 (96) 8.1 36 (97) 8.3 28 (100) 27(100) 22 (100)b 8.4 19 (100) 36(100) 13 (100)b

'logro Vero cell PFU/ml. oUnderscored values obtained by simultaneouglyfeeding three mosquito strains on same hamsters. ing, Hawaii, and Shanghai Ae. albopicfus and in Ae. aegyptf (Table 5). Virus was not isolated Fiji Ae. pseudoscutellarusare significantly lower from larval progeny. (P < 0.05) than those of Singapore and Sri DISCUSSION Lanka Ae. albopictusand Fiji Ae. aegypti. Three strains of Ae. albopictus(Shanghai Frzo, Among the speciesand strains tested,the four Shanghai Fz and Hawaii) were compared by strains of Ae. polynesiensiswere the most sus- feeding lots of each simultaneously on each of ceptible to RR virus infection per os.Based on five viremic hamsters (underscored values in an estimatedblood meal volume of 5 pl, inges- Table 3). Although infection rates in the three tion of 30 to 160 Vero cell PFU resulted in strains were quite similar, MIDi,o values could infection rates of83 to \00Voin the four strains. not be determined becausenone ofthe lots had We did not determine extrinsic incubation pe- infection rates below 50%. No significant differ- riods; however,Kay (1982) found that RR virus ence (P > 0.05) in infection rates was found proliferated quickly in Ae. uigilax infected per when the results were compared in Fisher's Ex- os. Virus was present in the salivary glands on act Test. day 2 postfeeding, initial transmission by bite Virus transmission data for infected mosqui- occurredon day 4, and maximum transmission toes that refed on suckling mice 13 to 21 days efficiency was reached after 10 to 13 days. following the infective feed are summarized in Therefore, we attempted virus transmission Table 4. Each speciesand strain tested trans- with mosquitoesthat had been incubated13 to mitted virus with the exception of Ae. polyne- 21 days. Virus transmission data for Ae. poly- siensisfrom Fiji and, in this case, the sample nesiensisare variable (Table 4); however,each sizewas small (n : 13).The data are inadequate strain exceptthe onefrom Fiji transmittedvirus. for a rigorous statistical analysis. This exception is probably due to the small A total of.4,718third and fourth instar larvae number of specimenstested. In general, our from the secondoviposition cycle ofpotentially results concerningAe. polynesiensrs confirm and infected females was tested in 39 pools for RR extendthose of Gubler (1981),who showedthat virus. Infection rates in parental femalesranged the Rarotonga and Samoa strains are efficient from 87 to 1007oin Ae. albopirtus, Ae. pseud,o- experimental vectors of RR virus. scutellaris,and Ae. polynesiensis,and 40 to 48% Susceptibility of the remaining species and Jounr.rr,oF THEAunnrcer Mosqumo Corrnol Assocrnrrorq VoL. 3, No. 2

Table 4. Ross River virus transmission rates amongAed,es (Stegonyin) mosquitoesinfected by feeding on viremic hamstersor suckling mice. Transmissionb Titer of in- Mosquito species Strain fective meal' Days incub. % Aed.es aegypti Frji 6.0 2l 15 53 6.6 2l 20 85 8.5' t4 11 55 Ae. pse u.doscutellar is Fiji 7.0 2I 23 57 2l 1 100 8.5' 1/ 90 Ae. polynesiensis Fiji 6.8 2l 50 8.5" 74 80 Rarotonga 6.9 t7 475 8.5' L4 40 Samoa 6.2 t7 4 100 8.3" l4 23 13 Tahiti 5.7 t7 10 40 6.6 27 2 100 Ae. albopictus Hawaii 6.0 21 25 76 7.6 27 20 85 Sri Lanka 5.3 l5 1 100 Singapore 6.2 t4 23 52 6.5 13 16 69 Shanghai Frzo 4.7 I4 683 5.0 T4 24 7l 5.2 T4 26 92 6.5 T4 47 92 " Titers marked are for suckling mice and are expressedin TCIDso/ml; all other titers are for hamsters and are expressedas logroVero cell PFU/ml. bTransmission rates are based only on the number of infected mosquitoesthat refed (n). Discrepancies betweenthese numbers and the number of infected mosquitoeslisted in Tables 2 and 3 are due to the fact that some mosquitoesdid not refeedduring the virus transmission attempts.

Table 5. History of mosquito progeny tested for transovarial transmission of Ross River virus. Parental females Titer of Infection No. refed infective rate for second No. oflarvae Mosquito species Strain meal' (%o\b oviposition tested Aed.esalbopicttu Hawaii 6.0 87.2 64 668 7.6 95.0 84 1,500 Ae. aegypti Frji 6.0 39.6 83 800 6.6 47.9 74 r,250 Ae. pseudoscutellaris Fiji 6.0 98.6 38 250 Ae. polynesiensis Ftji 6.8 100.0 22 250 " lo9'6 Vero cell PFU/ml oBased on samplesof 20 or more individuals tested from lots that survived the secondoviposition cycle and an extrinsic incubation period of 21 days. strains to per os infection with RR virus falls be equally susceptible to per os infection with into two groupings. Shanghai and Hawaii Ae. RR virus despite the great difference in their albopicttts and Fiji Ae. pseudoscuteLlariswerc colgnization history (Table 2). Unfortunately, highly susceptible,and Singaporeand Sri Lanka the Singaporeand Sri Lanka colonieswere lost, Ae. albopictusand Fiji Ae. aegypti were less so. and direct comparisons with these particular It shouldbe emphasizedthat only Shanghaiand strains are no longer possible.In any event, with Hawaii strains of Ae. albopictuswere compared the possible exception of the Sri Lanka Ae. directly by feeding them simultaneously on the albopictusstrain, each of the speciesand strains same viremic hamsters and that no significant tested could reasonably be expected to become differencein susceptibility was found when com- infected after feeding on viremic humans with parisonswere done in this manner.The Shang- titers comparableto those demonstratedduring hai strains (F126and F2 generations)appear to the Rarotongaoutbreak (Rosenet al. 1981).In JUNE 1987 Vrcton ColtrpnrnNcn op Asoss

general,Fiji Ae. aegypti andAe. pseu.d.oscutellarisUniversity, for providing subcolonies from their and the four strains of Ae. albopirtusused in the insectaries. virus transmission experiments (Table 4) were efficient transmitters of RR virus. REFERENCES CITED Our attempt to demonstrate transovarial transmission of RR virus by orally infected Aaskov. J. G., J. U. Mataika, G. W. Lawrence,V. and D. adults during the second oviposition cycle met Rabukawaqa,M. M. Tucker, J. A. R. Miles 1981.An epidemic of Ross River virus with negative results. Kay (1982) found 2/51 A. Dalglish. infection in Fiji, 1979. Am. J. Trop. Med. Hyg' female and,0/71 male progeny of seven intra- 30:1053-1059. thoracicallyinoculated female Ae. uigilaxto con- Austin, F. J., T. Maguire and J. A. R. Miles. 1979. tain RR virus. Apparently, this is the only evi- Ross River virus in Fiji and New Zealand. Dengue denceof transovarialtransmission of this virus. Newsletter, South Pacific Commission, New Cale- Aedes polynesiensis is one of the several donia,No. 7:73-14. closely related species in the subgenus Stego- Belkin, J. N. 1962. The mosquitoes of the South myia that make up the scutellaris group. The Pacific. Vol. 1. Univ. Calif. Press,Berkeley and Los majority of speciesin this group have been rec- Angeles,608 p. D. J. 1981.Transmission of RossRiver virus ognized from the South Pacific, where one or Gubler, by Aed,espolynesiensis and.Aedes aegypti. Am. J. more membersoccur on every major inhabited Trop. Med. Hyg. 30:1303-1306. island group exceptNew Caledonia,the Loyal- Kay, B. H. 1982.Three modesof transmission of Ross ties, and New Zealand (Belkin 1962).Aedes al- River virus by Aed.esured&m (Skuse). Aust. J. Exp. bopictus,sometimes considered to be a member Biol. Med Sci. 60:339-344. of the scutellaris group in the broad sense Kay, B. H., J. G. Carley,I. D. Fanningand C. Filippich. (Belkin 1962),does not occur in the South Pa- 1979.Quantitative studiesofthe vector competence cific but is widespreadin the Oriental and In- of Aedesaegypti, Cul.ex annulirostrus and other mos- domalayan regions and is well established in quitoes (Diptera: Culicidae) with Murray Valley en- other arboviruses. J. Hawaii. More recently, this species has been cephalitis and Queensland Med. Entomol. 16:59-66. reported in the United States from Memphis, Kay, B. J., J. A. R. Miles, D. J. Gubler and C. J. Tennessee(Reiter and Darsie 1984), Harris Mitchell. 1982. Vectors of Ross River virus: An County, Texas (Sprenger and Wuithiranyagool overview,p. 532-536. Proc. Int. Seminar. Viral Dis- 1986),and several southeasternstates and in easesin Southeast Asia and the Western Pacific. Brazil (Morbidity and Mortality Weekly Report Canberra. 1986).Vector competencestudies with the Har- Marshall, I. D. and J. A. R. Miles. 1984. Ross River ris County strain are in progress,and the results virus and epidemicpolyarthritis, p. 31-56. In: K. F. (Ed.), will be reported separately.Aedes pseu.doscutel- Harris Current topics in vector research.New York, Praeger Publ. laris is also a member of the scutellnris group Morbidity and Mortality Weekly Report. 1986.Aedes and is known only from the Fiji Islands. Aedes ahopi,ctus infestation-United States, Brazil. aegypti has a cosmotropical distribution and is MMWR 35:493-495. present in Hawaii and several island groups of Reiter, P. and R. F. Darsie, Jr. 7984.Aedes albopicttrs the South Pacific. Obviously, each of these spe- in Memphis, Tennessee(USA): An achievementof cies has the potential for playing a role in future modern transportation? Mosq. News 44:396-399. outbreaks of RR virus in the Pacific basin and, Rosen, L., D. J. Gubler and P. H. Bennett. 1981. in the caseof Ae. aegypti and Ae. albopictus,in Epidemic polyarthritis (Ross River) virus infection other areas as well. Knowledge concerning the in the Cook Islands. Am. J. Trop. Med. Hyg. vector competenceof these speciesand strains 30:1294-1302. Snedecor,G. W. and W. G. Cochran. 1967.Statistical for RR virus may be useful in developingcontrol methods. 6th ed. Iowa State Univ. Press, Ames., strategiesin the event of future outbreaks. 593p. Sprenger,D. and T. Wuithiranyagool. 1986.The dis- coveryand distribution of Aedesahopictus inHarris ACKNOWLEDGMENTS County,Texas. J. Am. Mosq. Control Assoc.2:217- 2r9. We thank the following personsfor collecting Tesh, R. B., D. C. Gajdusek, R. M. Garruto, J. H. mosquito strains in the field and providing sub- Cross and L. Rosen. 1975. The distribution and coloniesfor our use:Dr. D. A. Shroyer,Hawaii; prevalenceof group A arbovirus neutralizing anti- Mrs. N. A. Jayasekera,Sri Lanka; Mr. Ng Say bodiesamong human populations in southeastAsia Kiat, Singapore;and Mr. M. K. Tooheyand Ms. and the Pacific islands. Am. J. Trop. Med. Hyg. 24:664-675. M. S. Goettel,Fiji. We also are grateful to Pro- Tesh, R. B., R. G. Mclean, D. A. Shroyer, C. H. fessorLu Bao Lin, Institute of Microbiology and Calisher and L. Rosen. 1981.Ross River virus (To- Epidemiology,Beijing; Dr. M. Trpis, The Johns gaviridae: Alphavirus) infection (epidemic polyar- Hopkins University, School of Hygiene and thritis) in American Samoa. Trans. R. Soc. Trop. Public Health; and Dr. J. E. Freier,Notre Dame Med.75:426-43I.