Journal of the American Mosquito Control Association, l81@):ZB4-2g9.2OO2 Copyright @ 2002 by the American Mosquito Control Association, Inc.

VECTOR COMPETENCE OF THREE NORTH AMERICAN STRAINS OF AEDES ALBOPICZUS FOR WEST NILE VIRUS'

MICHAEL R. SARDELIS,',3 MICHAEL J. TURELL,{5 MONICA L. O'GUINN,4 RICHARD G. ANDRE'ENO DONALD R. ROBERTS'

ABSTRACT To evaluate the potential for North American (NA) Aezle.salbopictus to transmit West Nile virus (WN)' mosquito strains derived from 3 NA sources (Frederick County, Marylind, FRED strain; Cheverly, MD, CHEV strain; Chambers and Liberty counties, Texas, TAMU strain) were tested. These strains were tested along with a previously tested strain from a Hawaiian source (OAHU strain). Mosquitoes were fed on 2- to 3-day-old chickens previously inoculated with a New York strain (Crow 397-99) of WN. All of the NA strains were competent laboratory vectors of WN, with transmission rates of 36, 50, 83, and 92Vo for the FRED, CHEy OAHU' and TAMU strains, respectively. The extrinsic incubation period for WN in Ae. albopictus held at 26"C was estimated to be lo days. Based on efficiency of viral transmission, evidence of natural infection, bionomics, and distribution, Ae. albopictzs could be an important bridge vector of WN in the southeastern USA.

KEY WORDS Aedes albopictus, West Nile virus, vector competence, extrinsic incubation

INTRODUCTION Since discovery of Ae. albopictus in Memphis, TN (Reiter and Darsie 1984), and Houston. TX West Nile virus (WN) was reported for the lst (Sprenger and Wuithiranyagool 1986), in the 1980s, time in the Western Hemisphere in 1999 and caused this species has become established throughout encephalitis in humans in New york City and an most of the southeastern USA, extending epizootic in native and exotic avian populations as far north as New Jersey (Moore and Mitchell 1997). (Centers for Disease Control and prevention ICDCI Throughout its range in the USA, Ae. albopictus is 1999a, 1999b; Lancioui et al. 2000). Testing field- an important human pest, aggressively biting dur- collected mosquitoes for evidence of WN infection ing daylight hours close to its breeding sites. Ex- during 1999 and 2000 indicated that Culex pipiens perimental transmission studies indicate that it is a L. is the primary vector of this virus and that a competent vector of several flaviviruses (Mitchell number of additional species may be secondary or 1991). Although it is relarively refractory to infec- bridge vectors (CDC 1999b,2000). potential sec- tion with St. Louis encephalitis virus (Savage et al. ondary or bridge vector species from which mul- 1994), a study with a strain of WN from the our- tiple instances of WN infection were reported in- break of WN in New York in 1999 indicated that clude Culex restuans Theobald, Culex salinarius Ae. albopictus is a highly efficient laboratory vector Coquillett, Aedes vexans (Meigan), Ochlerotatus of WN (Turell et al. 2001). However. the latter japonicus japonicus (Theobald), Ochlerotatus tris- study used a long-colonized strain of Ae. eriatus (Say), Ochlerotatus trivittatzs (Coquillett), albopictus from Hawaii, and therefore may not represent what and Culiseta melanura (Coquillett). In September occurs in nature in regions of the USA where 2000, a single pool of Aedes albopictus (Skuse) WN is now enzootic. collected in southeastern Pennsylvania showed ev- To elucidate the potential role of Ae. idence of WN infection (CDC 2000). albopictus in the epidemiology of WN in the eastern USA, we conducted laboratory studies of the vector compe- I tence of 4 strains: The views of the authors do not necessarily reflect the 2 newly colonized ones from position of the Department of Defense or the Department Maryland where WN is considered enzootic, a of the Army. In conducting research with animals, the long-colonized Texan strain, and the Hawaiian investigators adhered to the Guide for the care and use of strain (OAHU) used by Turell et al. (2001). Addi- laboratory animals, as prepared by the Committee on tionally, studies were done to evaluate viral repli- Care and Use of Laboratory Animals of the Institute of cation and dissemination in these mosquitoes over Laboratory Animal Resources, National Research Council time. (NIH Publication 86-23, Revised 1996). The facilities are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. 2 MATERIALS AND METHODS Division of Tropical Public Health, Department of pre- ventive Medicine and Biometrics, Uniformed Services Mosquitoes: Three North American (NA) strains University of the Health Sciences, 4301 Jones Bridge of Ae. albopictus were evaluated for their ability Road, Bethesda. MD 20814. to I transmit WN. These included the Frederick (FRED) Present address: U.S. Army Medical Research Unit- Kenya, Unit 64109, APO AE 09831-4t09. strain, derived from eggs collected in Frederick 4 Department of Vector Assessment, Virotogy Divisron, County, Maryland, in July 1999 (Sardelis and Tur- U.S. Army Medical Research Institute of Infectious Dis- ell 20Ol); the Cheverly (CHEV) strain, derived eases, 1425 Porter Street, Frederick, MD 21702-5011_ from larvae collected from discarded tires in Chev- 5 To whom correspondence should be addressed. erly, Prince George's County, Maryland, in June

284 DucrNleen2002 Vecror. CoMpETENcEop Arots tnoptcrus 285

1999; and the Texas A&M University (TAMU) evaluate viral replication and dissemination over strain, derived from specimens collected in Cham- time, each strain of mosquito was fed on a single bers and Liberty counties, Texas, in 1987. Addi- chicken that had been inoculated with WN 48 h tionally, the OAHU strain of Ae. albopictus, de- earlier. Immediately after ingesting the infectious rived from specimens collected in Honolulu, HI, in blood meal, 5 mosquitoes of each strain were 1971, was evaluated. The generation tested was F3 killed, triturated, frozen, and assayed as described for FRED and CHEY and )F.o for TAMU and above. Handling and maintenance after bloodfeed- -+ OAHU. Larvae were reared at 26 l'C and a 16- ing for the remaining mosquitoes was as described h photoperiod and fed ground catfish chow above, except that samples of 10 mosquitoes of (AquaMax Pond Plus 3000, Purina Mills, Inc., St. each strain were killed, ground, and frozen for later Louis, MO). assay on days 1, 4,7, lO, 14,21, and 28 after Virus and virus assay: The WN strain (Crow bloodfeeding. 397-99) was isolated from a dead crow found in Statistics: The infection rate was calculated as the Bronx, New York City, NX during an epizootic the number of infected mosquitoes/total tested in 1999 (Turell et al. 2000) and had been passaged X 100. The dissemination rate was calculated as the once in Vero cell culture. Viral stock suspensions, number of mosquitoes with positive legs/total test- triturated mosquito suspensions, and chicken blood ed x100. The transmission rate was calculated as samples were tested for infectious virus by plaque the number of mosquitoes transmitting virus by bite assay on African green monkey kidney (Vero) cells (chickens were viremic when bled the day after the as described by Gargan et al. (1983), except that transmission attempt)/the number of mosquitoes the 2nd overlay, containing neutral red, was added that fed on these chickens. Infection, dissemination, by chi- 2 days after the lst overlay. and transmission rates were compared as appropriate and sig- Vector competence studies: Mosquitoes, 4 to 5 square or Fisher exact tests were determined at the 95Vo days old, were allowed to feed on a chicken (Gallus nificant differences level (SAS Institute Inc. 1999). A 1-way gallus L.) that had been inoculated subcutaneously confidence analysis of variance (ANOVA) was used to test for 24 or 48 h earlier with 0.1 ml of a suspension con- differences in the mean titer (log transformed) of taining l0a' plaque-forming units (PFU) of WN. WN ingested among the strains. The means (1og When most of the mosquitoes in a carton had fed transformed) from the study of viral replication in (:15 min), the chicken was transferred to a 2nd the bodies of infected mosquitoes were subjected carton containing a different strain of mosquitoes. to 2-way ANOVA to evaluate the main effects for This was repeated until all 4 strains of mosquitoes strain and day after oral exposure and the interac- had an opportunity to feed upon the same chicken. tion term. For terms found to be significant (ot : Immediately after mosquito feeding, a 0.1-ml blood 0.05) by ANOVA, the means were separated by sample was obtained from the jugular vein of each Duncan's multiple range test (SAS Institute Inc. chicken and diluted in 0.9 ml of diluent (lOVo heat- 1999). inactivated fetal bovine serum in Medium 199 (In- vitrogen, Carlsbad, CA) with Earle's salts, NaHCO3, and antibiotics) plus 10 units of heparin per milli- RESULTS viremia at the time of mos- liter to determine the All strains of Ae. albopictus were susceptible to quito feeding. Engorged mosquitoes were trans- infection with WN at both viral titers tested (Table ferred to 3.8-liter cartons with netting over the open 1). Susceptibility to infection for each strain signif- end and maintained in an incubator as described icantly increased with viral titer (1'> 5.4, df : I, above. Four days after the infectious blood meal, P < O.O2). Additionally, within viral titer levels, an oviposition substrate was added to each cage. susceptibility to infection was associated with strain To determine transmission rates, some of the tested (12 > 11.6, df : 3, P < 0.009). At the higher mosquitoes that had taken an infectious blood meal viral titer, infection rates of the TAMU (96Vo) and were individually allowed to refeed on a 2-day-old OAHU (937o) strains were significantly higher (X' chicken 13 days after the initial infectious blood > 10.0, df : l, P < 0.002) than infection rates of meal. Immediately after the transmission attempt, the FRED (56Vo) and CHEV (677o) strains. Dis- -2O"C the mosquitoes were killed by freezing at semination rates for all strains and at both viral dos- for -5 min and their legs and bodies were triturated es were O-ll%o lower than the corresponding in- -7O"C separately in 1 ml of diluent and frozen at fection rate. Transmission rates for mosquitoes until assayed for virus. Presence of virus in a mos- exposed to a chicken with a viremia of 1068 were quito's body indicated infection, whereas virus in significantly affected by strain (X' : 10.80, df: 3, the legs indicated that the mosquito had a dissem- P : 0.013), ranging from 36Vo for the FRED strain inated infection (Turell et al. 1984). Mosquitoes not to 92Vo for the TAMU strain. However, the reduced used in the transmission attempts were killed and efficiency was due to a reduced infection rate be- ground as described above on day 14 after the in- cause the percentage of infected mosquitoes that fectious blood meal. developed a disseminated infection and the per- Viral replication and dissemination studies: To centage of mosquitoes with a disseminated infec- 286 Jounnel or rHe AN{eRrcAN Moseurro CoNrnor- AssocIATtoN VoL. 18,No.4

oou^

dd9|, +FRED -.- + -I- hn..= CHEV TAMU OAHU A* - vYqa E,0 flO\ -* ^G:

- ^: n 9: AA il 1l o ! o. 7'o FFFFSS9E dd:; HI zzzz "ld J,d !2;< P 6.0 |!99n e7^ s.0 .t0 9t;;q: a Oro- oo\oOo\ ,E.fqT = 4.0 z E o! > :;.e'E S 3.0 ETi g I 2.0 X X >> .$P€ F <0.7 * N C..l C.l z :o3.1= 047tot42l .: -!.:.:. !trEE FFFFOo,O\o. ots-.-l Daysafter oral exposure B @;: zzzz9\qq Hd6ii J-lcic\ "b4:. asl9yl g; -+- -.FCHEV F =^-H FRED +TAMU +OAHU o \O O C-l c.r 8.0 OnO\€ a6,:- X th 7.O o O> t-6Ec S u.o y. \ .o-O 5.0 AA! !^XAnn E I: i E E : 4.0 g€tro 0a o+!i-al*01TnE$qTqq cnN S 3.0 oH > P r) r o' 3E)9g9EU3 -r^z s <'€ O\ 0O O\ * t- cn I 2.0 c.]o*h*\o60\ E9H> UaOo i-

{ ?vbo 047t0t4212E vF4 -€€.o x x >> Days after oral exposure N€€*O-or6 Sh\Otr-t :a €O\O\ Fig. l. Mean viral titers over time in the bodies (A) tttrtttl e : - .d _o 9q r)€€\tOFr)6l iolos+n00€ U =- U:. and legs (B) of infected Aedes albopictus after oral ex- .+ E F E'> posure to a West Nile virus-infected chicken with a vi- F- cn 6 O \O t-- \O a?) c'{$r)\or)\oo\ol !r fr .?:n remia of 1068 plaque-forming units per milliliter of blood E :, 2Z: and held at 26"C. FRED, Frederick County, Maryland, o d; X FH: ? I i 0.13). 'F3 i'ir 0 ! =E - E.: i l!.U3 From a chicken with a viremia of 106.8PFU/ml 'it of blood, approximately 10,.t infectious virions o x)a Y a.)-.lt-j FF3.9 c .!du- were ingested per mosquito (Fig. 1A). The strain o; ,; Is i iEEs of mosquito had no significant effect on the amount oc) gEE.j ;! i of virus ingested (ANOVA; F : : qo:- ' i! I tl 0.04; df 3,16; :1 ry!-€. P : 0.99). Two-way ANOVA revealed that the ^>' .^auda--_l main effect for strain proved to be nonsignificant HtsHF (F : 2.2; df : 3,140; P = 0.088) and that the main SFEEFE:€"Eo".EU.c-*>= ,Y'**,"I.'rE"! effect for day after oral exposure was significant (F 0 r'': bI - x =x =x o H> : trAt^EAo^ 6.= !.: !.E b.EE 126.7; df : 5,140; P < 0.001). The mean riters q.i -= EF:= EF o - - ,:>'"E,:>.v* aU AU atr k in the bodies by strain and day after oral exposure o 9- a .\)- a . F i " I o i€ E,9 x i,bir; >,bl are displayed in Figure 1A. Means separation tests .9?€:.HT€-: ; EgEEETE? o>!:o>!: ho=o=o!!oo showed that viral titers significantly increased be- '4! E-r)F E-rr+ N! il.i d: tween days 4,7, lO, and 14, but did not signifi- BYvegY:: .:E*E*E.9PE!, cantly increase from day 2I to 28. The interaction -tr9.1392!u'9.6 >J)^>Jr) Y X.; X.;: x i - between strain and day after oral exposure proved u)2z+L!14Z* 5&g8E&;8n to be nonsignificant (F = 0.5; df : 15,140; P : EV-VEV-VcESXgIsX XEKE 0.95). During this particular study, infection rates Drcrvern 2002 VEcroR CovpereNce oF AEDESALBoPtcrus 287 for the FRED, CHEV TAMU, and OAHU strains albopictus, analysis of the data suggested that some werc 42,59, 88, and 867o, resPectivelY. intraspeciflc variation may occur in vector compe- Evidence was found of virus escaping the midgut tence among NA strains of Ae. albopiclas for WN. of the FRED and OAHU strains 4 days after oral Previous studies have shown that vector compe- exposure (Fig. 1B). By 7 days after oral exposure, tence for flaviviruses varies widely among popu- virus was detected in the legs of all strains. Viral lations of Aedes (Gubler and Rosen 1976, Gubler titers in the legs of all strains peaked between l0 et al. 1979, Kay et al. 1984, Tabachnick et al. 1985, and 14 days after the infectious blood meal. For all Boromisa et al. 1987, Tian et al. 1999). Further the strains combined, the percentages of infected studies with more NA strains of Ae. albopictus mosquitoes that showed evidence of having a dis- from a wider range of geographic sources need to seminated infection (virus in the legs) on days 4, be done to elucidate this issue. 7, 10, 14, 21, and 28 were 14, 85, 100, 96, 100, Knowledge of the extrinsic incubation period arrd 96Vo, respectively. (EIP), along with other factors, is vital for estimat- ing vectorial capacity. The transmission studies showed that Ae. albopictus transmitted WN 13 days DISCUSSION after taking an infectious blood meal. Additionally, This study showed that the NA strains of Ae. the studies of viral replication and dissemination albopictus have the potential to serve as WN vec- over time indicated that virus had escaped the mid- tors based on their susceptibility to infection and gut by day 7 in >8OVo of all infected mosquitoes their ability to transmit WN efficiently. This finding and that the amount of virus circulating in the he- is consistent with previous laboratory transmission molymph (as estimated by the titer in a mosquitoes studies that showed that WN is transmitted by a legs) peaked between l0 and 14 days after the in- broad range of NA mosquito species, including a fectious blood meal. Although the presence of virus number of Culex and Aedes species (Turell et al. in the hemolymph does not indicate that the sali- 2000. 2001: Sardelis and Turell 2001; Sardelis et vary glands are infected and that infectious virions al. 2001). Additionally, the transmission rate for the are present in the saliva, it is probable that, based OAHU strain in this study (837o) closely agreed on the viral replication and dissemination studies, with the rate reported by Turell et al. (2001), who the EIP may be around 10 days. Additional trans- estimated the transmission rate for the same strain mission studies are needed to more precisely deter- to be 73Vo when tested under nearly identical con- mine the EIP and to evaluate the effect of temper- ditions. Because WN-infected wild birds (e.g., ature on EIP for WN in Ae. albopictus. The crows and house sparrows) can develop viremias duration of the EIP in this study is typical of the >108 PFU/ml of blood (Work et al. 1955), the vi- EIP of 9-12 days reported for other flavivirus-mos- remias of lOsr 68 PFU/ml used in our study should quito combinations at comparable incubation tem- be representative of what the mosquitoes would be peratures (Watts et al. 1987, Miller et al. 1989, Re- exposed to in nature. isen et al.1993). Of the barriers to the biological transmission of Although Ae. albopictu,t seems to be among the arboviruses by bite (midgut infection barrier [MIB], most efficient laboratory vectors of WN, a number midgut escaper barrier [MEB], and salivary gland of important aspects of the mosquito's bionomics barrier ISGBI), the MIB appeared to be the most must be considered to properly evaluate whether or important determinant of vector competence of the not this species will become important in the trans- strains tested in this study. However, by increasing mission of WN in nature. Aedes albopiclzs is con- the titer of the infectious blood meal, the MIB sidered an opportunistic feeder, taking blood meals could largely be overcome. Infection rates were from birds and mammals, including humans (Tem- identical or nearly identical to dissemination rates, pelis et al. 1970, Sullivan et al. l97l). Relatively thus indicating that a MEB was not important. Last- recent studies of host-seeking patterns of Ae. al- ly, because transmission rates were comparable bopictus in North America found that 3-l6Vo of its with dissemination rates, only a slight, if any, SGB blood meals are from birds (Savage et al. 1993, was apparent. Of the NA mosquito species tested Niebylski et al. 1994). The flight range of Ae. al- thus far (Turell et al. 2000, 2001; Sardelis and Tur- bopictus is relatively short, approximately a few ell 2OOl; Sardelis et al. 2001), laboratory vector hundred meters (Bonnet and Worcester 1946, Ro- efficiency is primarily modulated by a MIB. Ad- sen et al. 1976), which may limit its role in WN ditionally, these earlier studies indicate that vector transmission because of the need to become infect- competence level ranges widely, from inefficient ed at an avian focus of infection and then fly to (15Vo transrnission rate) through highly efficient human habitations. However, the mosquito's con- (>6OVo transmission rate), and that a pronounced tainer breeding sites are often found associated with MEB occurs in a number of common Culex spe- human habitation, and the avian reservoir hosts of cies. The Ae. albopictus strains tested here were WN (e.g., crows, blue jays, and house sparrows) efficient to highly efficient laboratory vectors of are ubiquitous. wN. Aedes albopictus is established throughout the Although we only evaluated a few strains of Ae. southeastern USA (Moore and Mitchell 1997) and 288 JounNer- oF THE AMERIcIN Mosqurro CoNrtoL AssocnrroN Vol. 18, No

is commonly reported to be an important biting date: West Nile virus activity---eastern United States, pest. Aedes albopictus has been found infected with 2OOO.Morb Mortal Wkly Rep 49:1O44-1O47. eastern equine encephalomyelitis virus (Mitchell et Centers for Disease Control and Prevention. 2001. Weekly al. 1992), a virus with a similar enzootic transmis- update: West Nile virus activity-United States, No- sion cycle to WN. In 2000, WN was detected in a vember 14-20,2001. Morb MortalWkly Rep 50:1061- 1062. single pool of Ae. albopictus captured in south- Gargan TP II, Bailey CL, Higbee GA, Gad A, El eastern Pennsylvania (CDC 2000), close to the Said S. 1983. The effect of laboratory colonization on the vec- northernmost established range of this species in tor pathogen interaction of Egyptian Culex pipiens and the mid-Atlantic region. West Nile virus was de- Rift Valley fever virus. Am J Trop Med Hyg 32:1154- tected in 4 Atlantic states (Connecticut, Maryland, I 163. New Jersey, and New York) in 1999: in 12 states Gubler DJ, Nalim S, Tan R, Saipan H, Sulianti Saroso J. and the District of Columbia, extending from most 1979. Variation in susceptibility to oral infection with of the New England states to North Carolina in dengue viruses among geographic strains of Aedes ae- 2000 (CDC 2000); and in 27 states and the District gypti. Am J Trop Med Hyg 28:1045-1O52. of Columbia in 2001 (CDC 2001). As WN contin- Gubler DJ, Rosen L. 1976. Variation among geographic ues its southward spread, the probability of contact strains of Aedes albopictus in susceptibility to infection with dengue between this pathogen and Ae. albopictus will be viruses. Am J Trop Med Hyg 25:318-325. Kay BH, Fanning ID, Carley JG. 1984. greater. As such, personnel involved in the ento- The vector com- petence of Australian Culex annuliro^srris with Munay mological arm of WN surveillance programs Valfey encephalitis and Kunjin viruses. Aust J Exp Biol should ensure that Ae. albopictus is collected and Med Sci 62:641-65O. tested. Also, further evidence of WN infection in Lanciotti RS, Roehrig J! Deubel V Smith J, Parker M, wild-caught A e. albopictus is needed to more firmly Steele K, Crise B, Volpe KE, Crabtree MB, Scherret incriminate this mosquito as a vector of WN. JH, Hall RA, MacKenzie JS, Cropp CB, Panigrahy B, The results of this study, combined with evi- Ostlund E, Schmitt B, Malkinson M, Banet C, Weiss- dence of natural infection in and knowledee of the man J, Komar N, Savage HM, Stone W, McNamara t distribution and bionomics of Ae. albopic'tus, sttg- Gubler DJ. 2000. Origin of the West Nile virus respon- gest that this species could function as a bridge vec- sible for an outbreak of encephalitis in the northeaitern tor for WN between the enzootic Culex spp.-avian United States. Science 286:2333-2337 . Miller BR, Mitchell cycle and susceptible mammalian hosts, including CJ, Ballinger ME. 1989. Replication, tissue tropisms and transmission of yellow humans. Because of the possibility of intraspecific fever virus in Aedes albopictus. Trans R Soc Trop Med variation in vector competence, Hyg 83: testing of local 252-255. strains of Ae. albopictus to determine their trans- Mitchell CJ. 1991 . Vector competence of North and South mission efficiency may be warranted to best esti- American strains of Aedes albopictus for certain arbo- mate the role this species may play in the epide- viruses: a review. J Am Mosq Control Assoc 7:446- miology of WN in a particular area. 45t. Mitchell CJ, Niebylski ML, Smith GC, Karabatsos N, Martin D, Mutebi ACKNOWLEDGMENTS JB Craig GB, Mahler MJ. 1992. Iso- lation of eastern equine encephalitis virus from Aedes We thank C. Bosio, D. Dohm, and K. Kenyon albopictus in Florida. Science 257:526-527. for their critical reading of the manuscript; J. Olson Moore CG, Mitchell CJ. 1997. 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