Isolation of West Nile Virus from Mosquitoes (Diptera: Culicidae) in the Florida Keys, Monroe County, Florida

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1 Caribbean Journal of Science, Vol. 40, No. 3, , 2004 Copyright 2004 College of Arts and Sciences University of Puerto Rico, Mayagüez Isolation of West Nile Virus from Mosquitoes (Diptera: Culicidae) in the Florida Keys, Monroe County, Florida LAWRENCE J. HRIBAR 1,LILLIAN M. STARK 2,ROBIN L. STONER 2,DAVID J. DEMAY 1, ANDREA L. NORDHOLT 1,MARIE J. HEMMEN 1,JOSHUA J. VLACH 1, AND EDSEL M. FUSSELL 1 1 Florida Keys Mosquito Control District, 5224 College Road, Key West, Florida Florida Department of Health, Bureau of Laboratories, Tampa Branch Laboratory, 3602 Spectrum Boulevard, Tampa, FL, Corresponding author: 1 lhribar@keysmosquito.org ABSTRACT. The detection of West Nile (WN) virus in the Florida Keys instigated an effort to determine which mosquito species might serve as vectors of this virus. Mosquitoes were collected in carbon dioxidebaited light traps and assayed for WN virus by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. A total of 53,673 mosquitoes in 3,643 pools (34 species) was assayed. Five species of mosquitoes were found positive for WN virus: Culex erraticus, Cx. nigripalpus, Cx. quinquefasciatus, Ochlerotatus condolescens, and Oc. taeniorhynchus. No significant difference was detected among the infection rates for the species analyzed in this study. KEYWORDS. Culicidae, Flaviviridae, West Nile virus, Florida Keys INTRODUCTION In July 2001, West Nile (WN) virus was detected in Florida. By the end of the year, WN virus activity had been observed in 65 of the 67 counties in the state; 1,106 wild birds, 492 horses, 194 sentinel chickens, 28 mosquito pools, and 12 humans were found infected with the virus. Human cases of WN virus were first detected in the Florida Keys, Monroe County, FL, late in 2001 (Blackmore et al. 2003). The review by Sampathkumar (2003) is a starting point for anyone unfamiliar with WN encephalitis. An intense mosquito surveillance program was put into action with the assistance of the Centers for Disease Control and Prevention. During the remainder of 2001 three mosquito species commonly collected in the Florida Keys, Anopheles atropos Dyar and Knab, Deinocerites cancer Theobald, and Ochlerotatus taeniorhynchus (Wiedemann), were positive for WN virus (Hribar et al. 2003). No positive mosquitoes were detected during 2002, although over 30,000 mosquito specimens were assayed for WN virus. This report describes isolations of WN virus from mosquitoes during MATERIALS AND METHODS Mosquito collections were made with carbon dioxide-baited ABC traps from January to December Collections were made from Big Coppitt Key, Big Pine Key, Boca Chica Key, Cudjoe Key, Cross Key, Fleming Key, Grassy Key, Indian Key, Key Largo, Key West, Lignumvitae Key, Long Key, Lower Matecumbe Key, No Name Key, Plantation Key, Ramrod Key, Sugarloaf Key, Stock Island, Upper Matecumbe Key, Vaca Key, and Windley Key. Traps were set in mid- to late afternoon. Mosquitoes were collected the following morning, taken to the laboratory, and frozen. Sorting and identification were done on a chill table, after which specimens were stored at 75 C. Mosquitoes were pooled by species, location, and collection date and shipped on dry ice to the Florida Department of Health Branch Laboratory in Tampa. Pools of mosquitoes were homogenized in 1 ml cell culture diluent using a Qiagen Mixer Mill. This medium was removed via Eppendorf 1000 L pipette, replaced by 1 ml lysis buffer, and homogenization was repeated. RNA was extracted from the lysate and assayed for WN by re-

2 WEST NILE VIRUS IN THE FLORIDA KEYS 363 verse transcriptase-polymerase chain reaction (RT-PCR) in an ABI Prism 7700 Sequence Detection system using TaqMan reagents. Primers and probe sequences were those from Lanciotti et al. (2000). The cell culture diluent was centrifuged, passed through a 0.22 m sterilizing filter that had been pretreated with fetal calf serum, and inoculated onto confluent monolayers of VERO cell culture in 25 cm 3 flasks. Details of methodology are available from Beaty et al. (1989). Cultures were observed periodically for 14 days postinoculation. RNA was extracted from cultures exhibiting a cytopathic effect and was assayed by RT-PCR as described above. Some specimens of Tabanidae and Ceratopogonidae were analyzed in the same manner as were mosquito pools. Infection rates (IR) per 1,000 for each mosquito species were calculated using the software developed by Biggerstaff (2003). This computer program resolves concerns over use of maximum likelihood estimation relative to minimum infection rates as discussed by Gu et al. (2003). Differences among IR were investigated by comparison of overlap of 83% confidence limits. This percentage confidence limit was chosen because it most closely approximates a statistical test of = 0.05 (Payton et al. 2003). RESULTS A total of 53,673 mosquitoes in 3,643 pools was collected during this study, comprising 34 species or species groups (Table 1). Five species of mosquitoes (Culex erraticus, Cx. nigripalpus, Cx. quinquefasciatus, Ochlerotatus condolescens, and Oc. taeniorhynchus) were found positive for WN virus during Eight positive pools were detected via molecular assay (MA) and two additional positive pools were detected via cell culture (VI) (Table 2). Examination of 83% confidence limits for IR revealed that all overlapped, indicating no significant difference among any of the IR for the species analyzed in this study. Three specimens of Tabanus lineola F. (Tabanidae), four specimens of Diachlorus ferrugatus (F.) (Tabanidae), and 10 specimens of an unidentified Culicoides sp. (Ceratopogonidae) were analyzed; all were negative for WN virus. DISCUSSION Ochlerotatus taeniorhynchus often is thought of as simply a nuisance mosquito, but it can be medically important (Lang 2003; Hribar et al. 2003). This species feeds primarily on mammals but will feed on birds (Edman 1971; O Meara and Edman 1975). Thus it can conceivably serve as a bird to mammal vector. Despite its relatively low susceptibility to infection with WN virus (Turell et al. 2001a), the detection of WN virus in five pools, the enormous numbers of Oc. taeniorhynchus that occur periodically in the Florida Keys (Hribar 2002), and that this species will feed on both mammals and birds suggest that this mosquito species may play an important role in transmitting WN virus from the avian cycle to mammalian hosts, such as humans and horses. Culex nigripalpus Theobald is believed to be one of the most important potential vectors of WN virus in Florida due to its association with Saint Louis encephalitis (SLE) virus and its life cycle (Turell et al. 2001b). It is an efficient laboratory vector of WN virus (Sardelis et al. 2001). This species feeds on avian hosts during winter and spring, then shifts to mammalian hosts during summer and fall (Edman and Taylor 1968). Bird species in at least 11 orders are utilized as blood meal sources in Florida whereas the mammals most often utilized are ruminants and rabbits. Humans are also bitten by Cx. nigripalpus. Feeding on hosts by female Cx. nigripalpus is opportunistic and depends upon the available vertebrates in the area (Edman 1974). This species is occasionally found around homes (Hribar et al. 2001, 2004), and is an incriminated vector of WN virus in other parts of the USA (Turell et al. 2001b). Culex quinquefasciatus Say may be an important vector of WN virus. It has been implicated as a vector of SLE virus (Chamberlain et al. 1959) and transmission of WN virus by the bite of this species has been demonstrated in the laboratory (Sardelis et al. 2001). This species takes blood meals

3 364 L. J. HRIBAR ET AL. TABLE 1. Mosquito pools collected in Monroe County, Florida, Species Number of specimens Number of pools Positive pools Infection rate (IR) 83% confidence limits Aedes aegypti (Linnaeus) Aedes spp Anopheles atropos Dyar & Knab 5, An. crucians complex An. grabhamii Theobald Culex atratus Theobald Cx. bahamensis Dyar & Knab 1, Culex declarator Dyar & Knab a Cx. erraticus (Dyar & Knab) 1, ± 1.28 Cx. iolambdis Dyar Cx. mulrennani Basham Cx. nigripalpus Theobald 2, ± 1.71 Cx. peccator Dyar & Knab Cx. pilosus (Dyar & Knab) Cx. quinquefasciatus Say ± 3.37 Cx. salinarius Coquillett Culex (Melanoconion) spp Culex spp Deinocerites cancer Theobald 8, Mansonia titillans (Walker) Ochlerotatus condolescens (Dyar & Knab) 1, ± 1.21 Oc. infirmatus (Dyar & Knab) Oc. sollicitans (Walker) Oc. taeniorhynchus (Wiedemann) 26, ± 1.75 Oc. tortilis (Theobald) 1, Oc. triseriatus (Say) Ochlerotatus spp Psorophora ciliata (Fabricius) Ps. columbiae (Dyar & Knab) Ps. johnstonii (Grabham) Ps. pygmaea (Theobald) Psorophora spp Uranotaenia lowii Theobald Unidentified Totals 53,673 3, a This is a species newly identified in Florida (Darsie and Shroyer in press). from avian and mammalian hosts, including humans (Cupp and Stokes 1973; Edman 1974). It is very common in the Florida Keys (Hribar 2002; Hribar et al. 2001, 2004) and occurs as far south as Argentina (Knight and Stone 1977). Ochlerotatus condolescens (Dyar & Knab) is a new addition to fauna of the USA (Darsie 2003). Very little is known about its bionomics, but this species bites humans (Arnell 1976). This species is easily confused with Oc. infirmatus (Dyar & Knab), a species also present in the Florida Keys (Hribar 2002). There is some disagreement about the distribution of Oc. condolescens: Arnell (1976) writes that it is restricted to Caribbean Islands, whereas Knight and Stone (1977) include records as far south as Argentina. Culex erraticus (Dyar & Knab) mainly feeds on birds but will feed on mammals, which may permit it to be involved in transmission of some arboviruses. Most of the birds fed upon by Cx. erraticus are wading birds (Ciconiiformes) rather than song birds (Passeriformes) and this may limit its role in transmission of arboviruses (Edman 1979). Data collected during the 2001 sea-

4 WEST NILE VIRUS IN THE FLORIDA KEYS 365 TABLE 2. Mosquito pools positive for West Nile virus in the Florida Keys, Species Island Date collected Number in pool Detection a Culex erraticus (Dyar & Knab) Key Largo 7/21/03 11 MA Cx. nigripalpus Theobald Boca Chica Key 5/9/03 13 MA Stock Island 6/24/03 1 MA Cx. quinquefasciatus Say Long Key 7/14/03 1 VI Ochlerotatus condolescens (Dyar & Knab) Key West 5/13/03 2 MA Oc. taeniorhynchus (Wiedemann) Stock Island 6/10/03 40 MA Key Largo 6/19/03 41 MA Key Largo 7/17/03 50 MA Big Ping Key 9/3/03 50 MA Monroe County 8/10/03 1 VI a MA, molecular assay; VI, cell culture. son appear to support this, since most of the birds found positive for WN virus were Passeriformes. No Ciconiiformes were found to be positive (Blackmore et al. 2003). Our collection of Culex declarator Dyar and Knab was a most unpleasant surprise, as this mosquito species was unknown from Florida until very recently (Darsie and Shroyer 2004). This mosquito has been found naturally infected with dog heartworm (Labarthe et al. 1998). It is a known vector of SLE virus in the Brazilian Amazon (Vasconcelos et al. 1991), and is suspected of involvement in a temporary increase in SLE virus infections during flooding caused by construction of the Tucuruí dam in Brazil (Dégallier et al. 1992). However, it is nearly refractory to infection with Venezuelan equine encephalitis virus (Turell 1999). Adults appear to be exophilic in their behavior (Taipe-Lagos and Natal 2003). In Brazil, this species will bite dogs, cats, and humans, and is collected most commonly during the summer months (Labarthe et al. 1998). Larvae were collected in a cavity in the ground at the base of a fallen angico branco tree (Piptadenia sp.) in Brazil (Forattini et al. 1998). The identification of this species casts some doubt on identity of some mosquito pools. Culex declarator can be easily confused with Cx. erraticus or Cx. nigripalpus (D. Shroyer, pers. comm.), and it is possible that our positive pool of Cx. erraticus may in fact be Cx. declarator. We hope that additional collections in 2004 will resolve this dilemma. Temperature plays an important role in determining whether mosquitoes can serve as efficient vectors of arboviruses (Dohm et al. 2002). The warm weather in the Florida Keys, where temperatures are usually in the seventies and eighties year round (Thomas 1974), is conducive to shortening the extrinsic incubation period for WN virus in mosquitoes. These same conditions and mosquito species should be of concern in other areas in or near the Caribbean, where WN virus already has been detected, viz., Mexico, the Dominican Republic, Jamaica, El Salvador, the Cayman Islands, and Guadaloupe (Estrada-Franco et al. 2003; Komar et al. 2003; Dupuis et al. 2003; International Society for Infectious Diseases 2001, 2003a,b; Pan American Health Organization 2003). LITERATURE CITED Arnell, J. H Mosquito studies (Diptera, Culicidae). XXXIII. A revision of the scapularis group of Aedes (Ochlerotatus). Contrib. Amer. Entomol. Inst. 13(3): Beaty, B. J., C. H. Calisher, and R. E. Shope Arboviruses. In Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections, eds. N. J. Schmidt and R. W. Emmons, Washington DC, American Public Health Association. Biggerstaff, B. J PooledInfRate: a Microsoft Excel Add-In to compute prevalence estimates from pooled samples. Centers for Disease Control and Prevention, Ft. Collins, Colorado. Blackmore, C. G. et al Surveillance results from the first West Nile virus transmission season in Florida, Am. J. Trop. Med. Hyg. 69:

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