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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Arboviruses encounter several potential infection and dissemination barriers within the mosquito, including the midgut and salivary gland infection barriers, and midgut and salivary gland escape barriers.5 Intrathoracic inoculations, which circumvent the midgut infection and escape barriers but not the salivary gland infection and escape barriers, ensure more uniform infection of some mosquitoes than oral exposure. Oral infection via artificial blood meals or viremic animals more closely resembles natural exposure. Several studies have demonstrated that mosquitoes are less susceptible to infection by artificial than by natural blood meals6–8 due to the concentration of virus adjacent to the midgut epithelium that follows natural but not artificial blood meals.9 In contrast, one study of Aedes aegypti mosquitoes infected with Semliki Forest virus found no difference in virus titers in infected mosquitoes after artificial versus natural blood meals except during the first 24 hours, and transmission did not differ significantly.10 However, infection rates, which usually are lowered by artificial blood meals compared with viremic animals, were not reported.
The use of viremic animals for assessing vector competence and transmission potential also has some disadvantages. Animal use in biomedical research is expensive, highly regulated, and requires special facilities. In addition, exposing mosquitoes with a predetermined dose requires knowledge about the animal’s viremia level after a given intrinsic incubation period. Variation in viremia responses among individuals can make the accurate prediction of viremia levels difficult. Also, good laboratory models may not exist because laboratory animals sometimes do not develop sufficient viremia levels to infect even proven vectors.
Many studies have estimated the arbovirus dose delivered by an infected mosquito during feeding.1,2,4,11–18 Chamberlain and others4 estimated the amount of eastern equine encephalitis virus (EEEV) inoculated by orally (viremic chicken) infected Ae. aegypti mosquitoes to be highly variable, ranging from undetectable to 5 log10 mouse intracerebral 50% lethal doses (ICLD50). A later study reported up to 3 log10 plaque-forming units (pfu) of EEEV in the saliva of Culiseta melanura deposited artificially into capillary tubes filled with immersion oil.18 A study of Semliki forest virus transmission by Anopheles albimanus using artificial blood feeding for both infection of mosquitoes and collection of saliva reported that 3.2 to 4 log10 mouse LD50 were transmitted.11 Using indirect methods, Mellink14 estimated that 2.7 log10 infectious doses of Venezuelan equine encephalitis virus (VEEV) per mg of saliva are transmitted by Ae. aegypti mosquitoes infected intrathoracically. Using quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) and capillary collection, Vanlandingham and others determined that Culex pipiens pipiens mosquitoes infected by an artificial blood meal contain an average of 4.3 log10 pfu of West Nile virus in their saliva with a range of 0.5 to 5.3 log10.17
Despite numerous studies cited above, important gaps remain in our knowledge regarding horizontal transmission of arboviruses. Different mosquito species have never been compared directly to determine if they transmit different amounts of a given virus. Different methods for virus exposure to mosquitoes have never been systematically evaluated for their effect on transmission and infectious doses delivered after an appropriate extrinsic incubation period. Also, the effect on transmission of the virus dose used to infect mosquitoes has not been evaluated. Additionally, the media used for saliva collection, oil and FBS, have not been compared. To address these issues, we used VEEV, an emerging arboviral pathogen of humans and equines,19 and two mosquito species: Aedes albopictus, a species that is susceptible to infection with VEEV and is often used as a model species for arbovirus transmission studies because it feeds readily in the laboratory,20,21 and Ochlerotatus taeniorhynchus, a proven VEEV vector.22–24 Three different routes for mosquito infection were compared along with two methods for collecting saliva. We determined the effect of mosquito species, route of infection, and the method of saliva collection on the amount of VEEV injected by mosquitoes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Viremia determinations. Six- to eight-week-old female NIH Swiss mice were injected subcutaneously (SC) with 1,000 pfu of VEEV. Three mice per time point were bled at 10, 14, 18, and 24 hours from the retroorbital sinus for viremia characterization. Serum titers were determined by plaque assay of serial 10-fold dilutions on Vero cell monolayers.
Mosquitoes. Ae. albopictus and Oc. taeniorhynchus mosquitoes were used for the reasons described above. Either F1 or F2 Ae. albopictus, derived from adult females collected in Galveston, Texas, or Oc. taeniorhynchus from a colony initiated with mosquitoes from Florida, were reared in an insectary at 27°C at 80% relative humidity using a light/dark cycle of 12:12 hours. Adult female mosquitoes were infected 6–8 days after emergence and incubated at 27°C for 5 or 10 days after infection, with 10% sucrose provided ad libitum during incubation.
Intrathoracic inoculations. Mosquitoes were inoculated in the thorax with approximately 1 µL containing 4 log10 pfu of VEEV using glass needles made from heated capillary pipettes.
Artificial blood meals. Mosquitoes were allowed to feed for 1 hour on an artificial blood meal containing 20% FBS, 1% sucrose, and 70% (V/V) packed sheep red blood cells and VEEV in Eagle minimal essential medium (MEM). Blood meal titers for each of three feedings were determined by plaque assay to be 6.1 and 8.1 log10 pfu/mL.
Viremic animal blood meals. Six- to eight-week-old female NIH Swiss mice were infected SC with 1,000 pfu of VEEV. Mice were anesthetized using pentobarbital and mosquitoes were allowed to feed for 1 hour. The mice were bled from the retroorbital sinus halfway through the blood meal to estimate the viremia titers of 6.1, 6.8, and 7.2 log10 pfu/mL. The University of Texas Medical Branch Institutional Animal Care and Use Committee approved all experiments with mice.
Saliva assays. Saliva was obtained by forced salivation into capillary tubes (10 µL capacity, VWR International, West Chester, PA) filled with either immersion oil (type B, Cargille Laboratories Inc., Cedar Grove, NJ) or 50% FBS/50% glycerol. The legs and wings were removed from individual mosquitoes, and the proboscis was inserted into a capillary tube containing the immersion oil or FBS/glycerol. Mosquitoes were allowed to salivate for 30–45 minutes, and salivation was confirmed in tubes containing immersion oil by the appearance of bubbles at the tip of the proboscis. Saliva could not be observed in tubes containing FBS/glycerol. Oil or FBS/glycerol and saliva were centrifuged into 100 µL of minimum essential medium (MEM) containing 20% FBS and frozen at –80°C until further processing; 30 µL were then added to monolayers of Vero cells and observed for cytopathic effects (CPE) for 5 days. Legs/wings and bodies were triturated separately in 300 µL of 20% MEM using a Mixer Mill 300 (Retsch, Inc., Newton, PA), and 75 µL of supernatant was added to Vero cells and observed for CPE for 5 days. All CPE-positive saliva samples were titrated by plaque assay on Vero cell monolayers.
Virus titration in mice. Intracerebral inoculation of 1- to 2-day-old mice was used to assay some saliva samples from mosquitoes positive for dissemination (infected legs) that had saliva negative for CPE, because it is more sensitive for detection of VEEV than cell culture-based methods.27 Twenty microliters of one 2.5-fold and three 10-fold serial dilutions were injected IC; mice that died were frozen at –80°C, and brains were assayed to confirm the presence of virus by CPE. The LD50 titers were calculated by the method of Reed and Muench.28
Statistics. Data that passed a normality test were analyzed by an unpaired t test. Data that were not normally distributed were analyzed by the Mann Whitney test or the Kruskal-Wallis test using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA).
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RESULTS |
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; Table 1). No significant difference (P > 0.05) occurred in virus detection rates or in saliva titers using immersion oil versus FBS; therefore, these groups were combined for further analysis.
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; Table 2), and the difference in saliva titers was no longer significant when compared with mosquitoes exposed by artificial blood meals or viremic animals. However, Oc. taeniorhynchus mosquitoes infected intrathoracically and incubated 5 days had higher saliva titers (P < 0.01) compared with those exposed to artificial blood meals or viremic animals (Figure 2; Table 2).
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; Table 2).
Effect of mosquito species on saliva titers.
For Ae. albopictus, the median cohort titers of virus in 208 saliva samples tested in cell culture ranged from 0.2 to 1.1 log10 pfu (mean range 0.7–1.4 log10 pfu), with a total range of 0.2 to 4.2 log10 pfu (Figure 2; Table 2). Overall, Oc. taeniorhynchus saliva contained larger amounts of VEEV than that of Ae. albopictus, with median titers of 0.2–3.2 log10 pfu (mean range 1.0–3.6 log10 pfu) and total range of 0.2–6.2 log10 pfu (Figure 2; Table 2). When individual cohorts were analyzed, the difference in saliva titers between mosquito species was significant for cohorts exposed intrathoracically (P < 0.0001) but not between cohorts exposed by artificial blood meals and viremic animals (P > 0.05).
Virus detection by inoculation of mice.
Intracerebral inoculation into suckling mice is more sensitive for detecting VEEV compared with vertebrate cell culture methods,27 as shown in Table 1. The limit of detection for our CPE assay using the volumes available was 0.5 log10 pfu; therefore, suck-ling mice were used for some saliva samples if the mosquitoes were positive for virus dissemination to the legs, but their saliva tested negative for CPE. Of 48 saliva samples from mosquitoes with a disseminated infection that had saliva negative by the CPE assay, 38 (79%) were positive by mouse inoculation, resulting in a 25% increase in sensitivity. The data gathered initially using mouse inoculation were not quantitative. Therefore, we determined the titers of saliva samples from Ae. albopictus mosquitoes by calculating suck-ling mice ICLD50 values for some saliva samples. Simultaneous titrations in Vero cells and mice indicated that the ICLD50: pfu ratio was approximately 200:1 (data not shown). Of 16 CPE-negative saliva samples from mosquitoes with disseminated infections, 3 tested positive in suckling mice and the ICLD50 values converted to pfu were 0.065, 0.054, and 0.065 log10 pfu. Five samples contained less than one mouse ICLD50 (less than 0.04 pfu). The addition of the mouse ICLD50 assay decreased the geometric mean titer of saliva by one tenth of a log10 pfu, and increased sensitivity by 2–31%.
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DISCUSSION |
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Effect of saliva collection medium. Our results indicate no difference in immersion oil versus FBS for the detection and quantification of VEEV in mosquito saliva. Silinized (to reduce possible virus adhesion) capillary tubes were also compared with non-silinized tubes spiked with VEEV, and no difference was noted (data not shown). The problems of contamination of the medium by mosquito mouthparts during in vitro collection of saliva, as discussed by Vanlandingham and others,17 were not experienced in our studies.
Effect of infection route on saliva titers.
Significantly less VEEV was detected in Ae. albopictus saliva after intrathoracic infection and a 10-day incubation period versus by an artificial blood meal and the same incubation. In contrast, when Ae. albopictus was infected intrathoracically and incubated 5 days, no significant difference in titers occurred compared with the artificial blood meal infection (Figure 2; Table 2). This difference in results with different incubation times should be assessed with other viruses in their mosquito vectors to determine if it represents a general phenomenon. The difference in Ae. albopictus is most likely due to different kinetics of viral replication in mosquitoes infected intrathoracically versus orally; VEEV titers peak earlier in intrathoracically infected mosquitoes because the midgut infection and escape barriers are circumvented. Our data suggest that when using intrathoracically infected mosquitoes, a shorter extrinsic incubation period should be used to generate peak saliva titers comparable to oral infection. Surprisingly, Oc. taeniorhynchus infected intrathoracically had a significantly higher mean saliva titer than those exposed via an artificial blood meal or viremic animal. Although mosquitoes can be less susceptible to infection using artificial blood meals compared with natural infection,6–8,29 our study found no difference in the dissemination rates observed after oral infection using the 2 methods, although there was a a 10-fold difference in the infectious dose.
Effect of infection dose on saliva titers.
Varying oral doses had a significant effect on Ae. albopictus saliva titers for mosquitoes exposed by artificial blood meal, but no difference was noted for mosquitoes exposed to varying oral doses from a viremic animal (Figure 2; Table 2).
Effect of mosquito species on saliva titers. The possibility that different mosquito species may transmit different amounts of virus has never been addressed. Our results demonstrate that saliva from Oc. taeniorhynchus mosquitoes contains on average 1.2 log10 pfu more VEEV than that of Ae. albopictus infected using the same methods and doses. Previous studies suggest that some epizootic VEEV strains undergo adaptation for Oc. taeniorhynchus transmission, and this adaptation may be a significant factor in epidemic emergence.25,30 The higher saliva titers in this proven vector, compared with Ae. albopictus that are susceptible to infection but have not been implicated in natural transmission, further support this hypothesis for VEEV emergence, although further studies examining the relationship of saliva titers to transmission efficiency are needed.
A wide range (0.2–4.2 log10 pfu) of VEEV titers was detected in the saliva of individual Ae. albopictus and Oc. taeniorhynchus (0.2–6.2 log10 pfu) mosquitoes, consistent with previous studies using other arboviruses.4,11,17 The amount of VEEV in the saliva did not correlate with the amount in the body or legs (data not shown). A previous study also showed no correlation between the amounts of dengue-2 virus transmitted by Ae. albopictus mosquitoes and the amounts of salivary gland tissue infected.13 The wide range of virus transmitted by mosquitoes is probably determined by factors other than the degree of salivary gland tissue infected after dissemination into the hemocoel.
Detection of low virus titers in saliva using mouse inoculation.
As seen in Table 1, a large number of mosquitoes was positive for disseminated infection (CPE-positive legs), but contained saliva that was negative by the CPE assay (31.4%). Inoculation of suckling mice demonstrated that small amounts of virus were present in many of these saliva samples, leaving only 6.5% samples negative for virus using both assay methods. The limit of detection for the CPE assay was 0.5 log10 pfu, but suckling mice inoculated IC proved to be much more sensitive than cell culture assays (Table 1).
An important remaining question is whether the amount of virus a mosquito salivates into a capillary tube accurately reflects the amount transmitted while feeding on a vertebrate host. To collect mosquito saliva in a capillary tube, the mosquito must first be cold anesthetized and have its legs and wings removed, traumatic events that may affect salivation. Additionally, mosquitoes are usually allowed to salivate into the capillary tubes for a much longer time than is required for engorgement on a host. To address these limitations of the current study, further experiments to assess amounts of VEEV transmitted in vivo are underway in our laboratory.
In conclusion, the VEEV titer used for the infection of mosquitoes appears to have little or no effect on the amount of virus found in saliva after extrinsic incubation. The method of oral infection (artificial blood meal versus viremic animal) also does not appear to affect the titer of VEEV in saliva. However, intrathoracic inoculation generates lower saliva titers when using the same incubation period (10 days). Ae. albopictus saliva (median titer of 0.6 log10 pfu, mean titer of 1.0 log10 pfu, range = 0.2 to 4.2 log10 pfu) contains significantly less VEEV than that of Oc. taeniorhynchus (median titer of 1.8 log10 pfu, mean titer of 2.2 log10 pfu, range = 0.2 to 6.2 log10 pfu), demonstrating that the mosquito species used in transmission studies can affect the quantity of virus transmitted and possibly the resultant pathogenesis. The results of this study should be considered when designing transmission and pathogenesis studies to mimic natural infection by arboviruses.
Received December 13, 2004. Accepted for publication February 8, 2005.
Acknowledgments: The authors thank Jing Huang for rearing mosquitoes and Lifang Zhang, James Grady, and Charles Fulhorst for statistical help. We also thank Lark Coffey, Todd Davis, and Nikolaos Vasilakis for kindly reviewing this manuscript and Slobodan Paessler for advice with animal work.
Financial support: This research was supported by NIH grants AI 418807 and AI 57156. D.R.S. was supported by the CDC training grant in Vector-Borne Infectious Diseases, T01/CCT622892. P.V.A. was supported by the James T. McLaughlin Fellowship fund. Authors’ addresses: Darci R. Smith, Anne-Sophie Carrara, and Scott C. Weaver, Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609, Telephone: 409-747-2440, Fax: 409-747-2415. Patricia V. Aguilar, Department of Microbiology and Immunology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1019, Telephone: 409-747-2440, Fax: 409-747-2415.
* Address correspondence to Scott C. Weaver, Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0609. E-mail: sweaver{at}utmb.edu 
Reprint requests: Scott C. Weaver, Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609, Telephone: 409-747-2440, Fax: 409-747-2415, E-mail: sweaver{at}utmb.edu.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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