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    Everglades virus transmission cycle. Arrows indicate cycling of Everglades virus via mosquito vectors and vertebrate reservoir hosts. Organisms with an established role in the cycle are highlighted in bold; those species postulated to participate are shown in normal type.

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    EVEV viremia in adult female NIH Swiss mice. Bars indicate standard deviations.

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SUSCEPTIBILITY OF OCHLEROTATUS TAENIORHYNCHUS AND CULEX NIGRIPALPUS FOR EVERGLADES VIRUS

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  • 1 Department of Pathology and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas

Everglades virus (EVEV), an alphavirus in the Venezuelan equine encephalitis (VEE) complex, is a mosquito-borne human pathogen endemic to South Florida. Field isolations of EVEV from Culex (Melanoconion) cedecei and laboratory susceptibility experiments established this species as its primary vector. However, isolates of EVEV from Ochlerotatus taeniorhynchus and Culex nigripalpus, more abundant and widespread species in South Florida, suggested that they also transmit EVEV and could infect many people. We performed susceptibility experiments with F1 generation Oc. taeniorhynchus and Cx. nigripalpus to evaluate their permissiveness to EVEV infection. In contrast to the high degree of susceptibility of Cx. (Mel.) cedecei, Oc. taeniorhynchus and Cx. nigripalpus were relatively refractory to oral EVEV infection, indicating that they are probably not important vectors. Identification of vectors involved in enzootic EVEV transmission will assist in understanding potential changes in vector use that could accompany the emergence of epizootic or epidemic EVEV.

INTRODUCTION

Everglades virus (EVEV), a member of the Venezuelan equine encephalitis (VEE) complex of alphaviruses (Togaviridae: Alphavirus), is a human pathogen endemic to South Florida that causes febrile illness and sometimes severe neurologic disease.13 Everglades virus is transmitted among reservoir rodents by mosquito vectors (Figure 1) in the Everglades and surrounding regions of South Florida, in close proximity to the Miami-Dade metropolitan area.4,5 This region is inhabited by more than 2 million people and hosts more than 1 million visitors annually,6,7 suggesting the potential for large-scale epidemic disease should EVEV emerge like related VEE complex viruses.

VEE virus (VEEV) emergence depends on a combination of viral mutation, epidemiologic, and ecological events that must coincide in time and space. The close relationship of EVEV to VEEV suggests that adaptation to equine amplification hosts or mosquito vectors, as has been observed in VEEV epizootics and epidemics, could likewise mediate EVEV emergence. Although EVEV is similar in many respects to other enzootic VEE complex viruses, there is no evidence that it has emerged to produce epidemics and equine epizootics as have some other enzootic strains. Despite the lack of evidence of EVEV emergence, suggesting that it does not pose a major public health risk, the recent emergence of VEE in Mexico despite no previous history of subtype IE epizootics indicates that emergence potential of VEE complex viruses is not entirely predictable.8

The absence of EVEV emergence could be explained by the lack of certain crucial ecological components of epizootic transmission in Florida.9 For example, the absence of large equine populations in South Florida10 may preclude efficient epizootic amplification. Extensive equine vaccination against alphaviruses or natural, cross-protective immunity are other possibilities.

Other possible explanations for the lack of EVEV emergence are related to the intrinsic amplification competence of the virus and its potential for adaptation to new hosts. VEEV epidemics are believed to arise when mutations in enzootic subtype ID strains generate epizootic variants that produce high-titered viremias in horses, resulting in amplification and spillover to humans.11 EVEV, which generates only low-titer viremias in experimentally infected horses,12 may not be capable of adapting for equine replication like the subtype ID strains due to fundamental genetic differences. In other words, EVEV may be incapable of mutating to produce an epizootic, equine amplification-competent phenotype.

Limitations in vector infectivity and transmission potential could also explain the absence of EVEV emergence. Epizootic VEEV cycles involve several different mammalophilic mosquito vectors,9,13 and adaptation to efficiently infect certain epizootic vectors such as Ochlerotatus taeniorhynchus may mediate efficient transmission to horses and people.14,15 EVEV may not efficiently infect vectors with the potential for widespread epidemic transmission and/or may not have the ability to readily adapt to these species. Further studies of EVEV-vector relationships could therefore improve understanding of the factors that limit EVEV transmission to humans and affect the potential for epidemic emergence and could assist in the development and implementation of strategies to prevent potential disease.

Everglades virus vectors.

Previous studies implicated Culex (Melanoconion) cedecei as the primary EVEV vector based on high infection rates in the field4 and its efficiency in transmitting EVEV to naïve animals in laboratory experiments.16 Of the 7 vectors of enzootic VEE complex viruses identified to date, all are members of the Spissipes section in the Culex (Melanoconion) subgenus.13

Relatively little is known about the potential for mosquitoes other than Cx. cedecei to vector EVEV in the enzootic cycle. Although most studies of enzootic South and Central American VEE complex virus ecology have incriminated a single Culex (Melanoconion) species as the principal vector at a given locality, three different mosquito species were implicated at a single forest in Colombia.17 Although evidence supports the role of Cx. cedecei as the principal enzootic EVEV vector, numerous isolates of EVEV from other mosquito species suggest that additional mosquitoes may also play a role in enzootic/endemic transmission. In field studies, 97 EVEV isolates were made from Ochlerotatus (previously Aedes) taeniorhynchus and 6 were made from Culex nigripalpus, and both species had minimum infection rates comparable to those of Cx. cedecei.4 Ochlerotatus taeniorhynchus has also been implicated as an important vector of epizootic/epidemic VEE complex viruses in South and Central America.1822 However, because the EVEV isolates made from Oc. taeniorhynchus and Cx. nigripalpus may have been present only in viremic blood-meals or in nondisseminated infections limited to the midgut, the potential for Oc. taeniorhynchus and Cx. nigripalpus to serve as EVEV vectors remained to be tested using susceptibility experiments.

Vector incrimination.

Traditionally, incrimination of arthropods as vectors of viral pathogens involves four criteria: 1) repeated demonstration of natural infection, 2) demonstration of effective contact between the virus and vector, 3) establishment of a temporal and spatial association between the virus and vector, and 4) validation of laboratory susceptibility and ability to transmit.23 In addition to fulfilling criterion 1,4 Oc. taeniorhynchus and Cx. nigripalpus also possess host feeding preferences and behavior conducive to transmitting EVEV (criterion 2). Ochlerotatus taeniorhynchus is an aggressive feeder and seeks both small vertebrates including rodent reservoirs and humans for blood,24,25 and Cx. nigripalpus is an opportunistic feeder on both mammals, including rodent reservoirs, and birds.26 Like Cx. cedecei, Oc. taeniorhynchus and Cx. nigripalpus exhibit crepuscular feeding periodicity,2426 which coincides with peak activity of rodent reservoirs (criterion 3). Both species are also capable of flying long distances24,26; a mosquito infected in an enzootic focus within several kilometers of residential locations could fly into highly populated areas and transmit EVEV to humans. Although Oc. taeniorhynchus and Cx. nigripalpus were implicated as EVEV vectors based on the first three criteria for vector incrimination, laboratory susceptibility must be demonstrated to definitively incriminate these species.

If EVEV is vectored by Oc. taeniorhynchus and Cx. nigripalpus, humans may be extensively exposed to this pathogen because these species are ubiquitous and abundant throughout South Florida.27,28 Even if these two species prove relatively insusceptible to EVEV, as indicated by low levels of virus infectivity and dissemination after ingestion of relevant oral doses of virus, their extremely high densities in EVEV endemic areas could still allow for a considerable amount of human exposure to EVEV-infected mosquitoes. Several marginally susceptible mosquito species have been implicated as VEEV vectors during epizootics when their large population sizes allow for transmission.13,29,30 As a related example, high densities of Aedes aegypti that were relatively resistant to infection with yellow fever virus were sufficient to initiate and maintain an urban epidemic in Nigeria.31 Alternatively, a lack of susceptibility of Oc. taeniorhynchus and Cx. nigripalpus may help explain the absence of recorded EVEV outbreaks. Therefore, we tested the susceptibility of these mosquitoes from South Florida to EVEV infection.

MATERIALS AND METHODS

Mosquito collection, identification, and rearing.

Adult mosquitoes were collected in CDC light traps (John W. Hock Company, Gainesville, FL) baited with dry ice in Mahogany Hammock, Everglades National Park (ENP) (25°20′24″N, 080°49′22″W), Florida, in June 2003. Progeny derived directly from natural populations were used to avoid changes in susceptibility to virus infection that might result from colonization and because Cx. nigripalpus mating is only successful under special circumstances in large outdoor cages.32 Mosquitoes were chilled and identified using morphologic criteria33 and were transported to an insectary at the University of Texas Medical Branch, Galveston, Texas. Females of each species were placed separately in 30 cm3 rearing cages at 27°C with a relative humidity of 80% and a 12 hour:12 hour (light: dark) photoperiod. To detect natural infection with arboviruses including EVEV, wild mosquitoes were offered blood meals from sentinel mice lacking functional interferon receptors, which uniformly die when infected with most or all arboviruses endemic to South Florida including VEE complex viruses.34 Later, mosquitoes were supplied with hamster blood-meals at weekly intervals and 5% sucrose water ad libitum. A pan of fresh water mixed with decomposing grasses was provided as an oviposition substrate for Cx. nigripalpus, and Oc. taeniorhynchus received a similar pan with a 1:1 (sea:fresh) water mixture and moss. First-generation females 5–7 days of age were used for all susceptibility experiments.

Viruses.

Two EVEV strains from ENP were used for mosquito susceptibility experiments: the prototype strain, FE-37c, isolated in 1963 from Culex (Melanoconion) spp. mosquitoes, was passaged five times in suckling mouse brains (SMB) and twice in Vero cells and has been fully sequenced35; strain FE4-71k (SMB1, Vero 1) is a 1964 isolate from a pool of 14 unfed Culex spp. mosquitoes. Both isolates were used to assess strain variation and to determine if the more extensive passage history of FE-37c altered its infection phenotype. A VEEV subtype IC epizootic strain, 3908, which had been passaged once in Vero cells, was also used as a positive control because Oc. taeniorhynchus are highly susceptible to infection with this epizootic subtype.19,21 Virus stocks were prepared in Vero cells and used for inoculation of mice.

Mosquito susceptibility experiments.

Mosquitoes were offered blood meals at natural crepuscular feeding times from viremic adult NIH Swiss mice that had been anesthetized with sodium pentobarbital. Manipulations of mice were carried out using a protocol approved by the University of Texas Medical Branch IACUC and were performed in accordance with recommended guidelines.36

Adult female NIH Swiss mice inoculated subcutaneously in the left thigh with approximately 4,000 PFU of EVEV developed peak viremias 24 hours postinoculation (Figure 2); mosquitoes were all fed from mice at this time to maximize the amount of ingested virus. Although there are no data in the literature indicating that the animal source affects the infectiousness of viremic blood of a given viral titer, we conducted experiments to compare blood meals from hamsters and mice to ensure that our results with mice could be compared directly with data published previously using hamsters. After engorging on mice or hamsters circulating similar viremia titers of VEEV strain 3908 or EVEV strain FE-37c, infection and dissemination rates in Oc. taeniorhynchus were not significantly different between rodent species (because viremias cannot be exactly duplicated for each feeding trial, titers varied slightly; see Table 2). Therefore, we used mice for all experiments because their peak viremias are comparable to levels observed in cotton rat reservoirs.37

For each feeding trial, one viremic mouse was presented to a cohort of 100 mosquitoes for approximately 1–2 hours. After the feed, mice were bled, and standard plaque assays38 were performed to determine mouse viremias at the time of feeding. Briefly, mouse sera were serially 10-fold diluted in phosphate buffered saline and 250 μL of each dilution was overlaid on confluent monolayers of African Green Monkey Kidney (Vero) cells grown in 6-well plates (Corning, Acton, MA). After a 1-hour incubation at 37°C, wells were overlaid with 4 mL of 1% agarose in Eagle’s minimum essential medium (MEM) and plates were incubated at 37°C to allow plaque development. To compare the Vero cell virus assay system used here to the CEC assay system used for previous Cx. cedecei experiments,16 we performed plaque assays on confluent monolayers of both cell types with the same stock inocula of both EVEV strains, as well as epizootic VEEV strain 3908. The CEC cells were first passage from embryo harvest (Spafas, Charles River Laboratories, Inc., Wilmington, MA). Two-tailed Student’s t tests39 were performed to evaluate whether significant differences in plaque assay titers exist between cell types.

Fully engorged mosquitoes were incubated in 0.5 l containers with 5% sucrose for a 12-day extrinsic incubation. Then, mosquitoes were killed and bodies and legs were individually homogenized (Retsch MM300 homogenizer, Retsch Inc., Newton, PA) in MEM with 20% fetal bovine serum and frozen at −80°C until virus assay. Infection was determined by recovery of virus from the homogenized body and dissemination was determined by recovery of virus from the legs of a given mosquito.40 The infection rate was recorded as the fraction of virus-positive bodies divided by the total number of bodies, and the dissemination rate is the number of virus-positive legs divided by the number of virus-positive bodies.

RESULTS

Table 1 compares virus titers in different cell types for EVEV and VEEV strains. There were no significant differences in plaque titers between cell types, indicating that one EVEV Vero cell PFU corresponds to one CEC PFU. These results, combined with data (Table 2) showing no difference in mosquito infectability using mice or hamsters as viremic hosts, confirm that the viremias in mice we presented to Cx. nigripalpus and Oc. taeniorhynchus were similar to levels ingested from hamsters by Cx. cedecei in previous studies.16

Results from the EVEV mosquito susceptibility experiments are shown in Table 3. Data from previous studies using Cx. cedecei16 are included for comparison. Viremia titers in EVEV-infected mice ranged from 3.8 to 5.5 Vero log10 PFU/mL at the time of mosquito feeding. Thirty-eight percent of the Oc. taeniorhynchus fed on strain FE-37c–infected mice became infected, but none of those mosquitoes developed disseminated (leg) infections. Similarly, only 29% of Oc. taeniorhynchus that fed on strain FE4-71k–infected mice became infected. Infection rates were not higher in cohorts of Oc. taeniorhynchus that ingested blood from mice with higher viremias. Of 86 Cx. nigripalpus that fed on viremic strain FE-37c–infected mice, only 2 became infected and neither of those mosquitoes contained detectable virus in their legs, indicating that they did not have virus dissemination into the hemocoel and could not transmit.

Unlike the results with EVEV, 90% of Oc. taeniorhynchus fed on a VEEV strain 3908-infected mouse with 8.3 Vero log10 PFU/mL became infected and 95% of the infected mosquitoes contained disseminated virus in leg assays; with a lower virus dose ingested (6.9 Vero log10 PFU/mL), 96% became infected and 82% developed disseminated infections, demonstrating that this Oc. taeniorhynchus population was susceptible to VEEV like others tested previously.14,15,19,21

DISCUSSION

Mosquito susceptibility to EVEV.

Our infectivity results for Oc. taeniorhynchus and Cx. nigripalpus contrast sharply with those from similar experiments performed with other mosquito species and VEE complex viruses. Eighty percent of Cx. cedecei became infected after ingesting a lower EVEV dose (3.6 log10 CEC PFU/mL) of viremic hamster blood and 100% were infected after ingesting blood from a hamster with a viremia of 4.3 log10 CEC PFU/mL.16 Unlike those exposed to EVEV, 90% or more of Oc. taeniorhynchus that ingested a VEEV epizootic strain from a viremic mouse became infected, and the majority contained disseminated virus in leg assays, consistent with previous studies indicating high susceptibility to epizootic VEEV.14,15,19,21

The susceptibility of a mosquito to oral infection and its ability to support virus dissemination are among the primary determinants of vector incrimination. Despite ingesting doses of virus from viremic mice that correspond to peak viremias of rodent reservoirs (cotton rat viremias peak at approximately 4.0 log10 Vero PFU/mL 1–2 days postinoculation, 37 Oc. taeniorhynchus and Cx. nigripalpus from Florida rarely became infected with EVEV and none developed disseminated infections required for transmission. The possibility that natural transovarial EVEV transmission was responsible for the small number of infections we observed is highly unlikely because we detected no transmission to interferon receptor–deficient mice used to feed the field-collected mosquitoes, and vertical transmission of VEEV was not documented in more than 5,000 larvae reared from infected female mosquitoes.41 The absence of EVEV dissemination in both Oc. taeniorhynchus and Cx. nigripalpus suggests that these mosquitoes exhibit midgut escape barriers. Although relatively insusceptible mosquitoes have served as arbovirus vectors in the past,31 the complete lack of disseminated infections in the mosquitoes we tested indicates that these species are incapable of transmission.

Our results indicate that the high minimum infection rates observed in field collected Oc. taeniorhynchus and Cx. nigripalpus,4 similar to levels for Cx. cedecei, probably represent EVEV uninfected mosquitoes that had recently ingested viremic blood meals or mosquitoes with nondisseminated infections limited to the midgut, without the ability to transmit. These results demonstrate that Oc. taeniorhynchus and Cx. nigripalpus are not efficient EVEV vectors and probably do not play an important role in enzootic EVEV transmission.

Specificity of VEE complex viruses for enzootic vectors.

Our findings are consistent with the view that enzootic VEE complex viruses are highly specific in their use of Culex (Melanoconion) mosquitoes as vectors and that other species do not play important roles in enzootic transmission cycles.13 Similar to our observations and consistent with this pattern of vector use, Oc. taeniorhynchus are less susceptible to infection with an enzootic Central and South American subtype ID and IE VEEV than to infection with an epizootic/epidemic subtype IC strain of VEEV,14,18,22,42 and Cx. nigripalpus are refractory to infection with enzootic subtype IE VEEV.42

Potential for EVEV emergence.

Our results have important implications for understanding EVEV transmission dynamics, defining the virus transmission cycle, and mitigating disease, should outbreaks of EVEV occur. The insusceptibility of Oc. taeniorhynchus and Cx. nigripalpus, two abundant, widespread mammalophilic species, may explain why outbreaks of EVEV have not occurred in South Florida. Previous studies43,44 indicate that epidemic/epizootic VEEV emerge from subtype ID strains, the closest relatives of EVEV. Such emergence events arise when combinations of mutations that alter equine virulence and/or mosquito infectivity occur in concert with appropriate ecological conditions. Genetic studies suggest that only a few mutations in enzootic VEEV can generate viruses with epizootic (equine amplification and/or epizootic vector-competent) phenotypes.11,14 These epizootic phenotypes are mediated principally by the E2 envelope glycoprotein that is believed to interact with cellular receptors.14,45 For related enzootic VEEV in Mexico, a single amino acid substitution in the E2 glycoprotein dramatically increases the Oc. taeniorhynchus infectivity phenotype.15

Studies to examine the potential for EVEV to generate an epizootic/epidemic phenotype via E2 mutations and its ability to change its vector host range are underway in our laboratory. If outbreak strains of EVEV arise, infection of equines or humans living in South Florida could cause substantial morbidity or mortality. Delineating the determinants of vector susceptibility that affect the transmission dynamics of EVEV may be important for the development and implementation of strategies to prevent potential epidemic disease.

Table 1

Comparison of plaque titers of EVEV and VEEV strains in African green monkey kidney (Vero) and chicken embryo (CEC) cell assays

Mean titer ± standard deviation (log10 PFU/mL)
Virus strainVeroCECP value
EVEV FE-37c8.6 ± 0.198.5 ± 0.080.50
EVEV FE4-71k8.3 ± 0.348.6 ± 0.120.32
VEEV 39088.7 ± 0.188.6 ± 0.190.06
Table 2

Comparison of infection and dissemination rates in Ochlerotatus taeniorhynchus feeding on viremic mice or hamsters

Rodent species*Virus strainViremia titer (log10 PFU/mL)Fraction infected (%)Fraction infected with dissemination (virus in legs) (%)†
* NIH Swiss mice and golden Syrian hamsters used in experiments were adult females.
Ochlerotatus taeniorhynchus used for this preliminary experiment were from a colony of mosquitos originating from Florida.
MouseEVEV
 FE3-7c5.115/71 (21)1/15 (7)
HamsterEVEV
 FE3-7c4.02/25 (8)0/2 (0)
MouseVEEV
 39086.971/78 (91)57/71 (80)
HamsterVEEV
 39085.628/29 (97)24/28 (86)
Table 3

Infection and dissemination rates in Oc. taeniorhynchus, Cx. nigripalpus, and Cx. cedecei fed on EVEV-infected mice

Mosquito speciesVirus strainViremia titer (log10 PFU/mL)Fraction infected (%)Fraction infected with dissemination (virus in legs) (%)
NA, not applicable.
* Data published previously16; Cx. cedecei mosquitoes (F20–F30) aged < 14 days from the same location were fed on viremic hamsters and incubated 19–22 days. Three out of three infected Cx. cedecei also transmitted EVEV to a naïve hamster.
Cx. cedecei*EVEV FE-37c3.64/5 (80)Not reported
4.313/13 (100)Not reported
Oc. taeniorhynchusEVEV FE-37c4.76/10 (60)0/6 (0)
5.52/2 (100)0/2 (0)
3.84/16 (25)0/4 (0)
4.813/37 (35)0/13 (0)
Oc. taeniorhynchusEVEV FE-37cOverall25/65 (38)0/25 (0)
Oc. taeniorhynchusEVEV FE4-71k4.52/7 (29)0/2 (0)
Oc. taeniorhynchusVEE 39088.319/21 (90)18/19 (95)
Epizootic6.971/74 (96)58/71 (82)
Cx. nigripalpusEVEV FE-37c5.20/28 (0)NA
5.52/4 (50)0/2 (0)
4.90/27 (0)NA
4.50/27 (0)NA
Cx. nigripalpusEVEV FE-37cOverall2/86 (2)0/2 (0)
Figure 1.
Figure 1.

Everglades virus transmission cycle. Arrows indicate cycling of Everglades virus via mosquito vectors and vertebrate reservoir hosts. Organisms with an established role in the cycle are highlighted in bold; those species postulated to participate are shown in normal type.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 1; 10.4269/ajtmh.2005.73.1.0730011

Figure 2.
Figure 2.

EVEV viremia in adult female NIH Swiss mice. Bars indicate standard deviations.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 1; 10.4269/ajtmh.2005.73.1.0730011

*

Address correspondence to Lark L. Coffey, Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609. E-mail: llcoffey@utmb.edu

Authors’ addresses: Lark L. Coffey and Scott C. Weaver, Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0609, Telephone: (409) 747-0758 (Weaver), (409) 747-2440 (Coffey), Fax: (409) 747-2415.

Financial support: L.L.C. was supported by the James W. McLaughlin Fellowship Fund. This research was supported by NIH grant 48807.

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Author Notes

Reprint requests: Scott Weaver, Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0609.
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