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Am. J. Trop. Med. Hyg., 77(1), 2007, pp. 176-187
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene

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Infection and Dissemination of Venezuelan Equine Encephalitis Virus in the Epidemic Mosquito Vector, Aedes taeniorhynchus

Darci R. Smith*, Nicole C. Arrigo, Grace Leal, Linda E. Muehlberger, AND Scott C. Weaver
Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, Histopathology Service Core, University of Texas Medical Branch, Galveston, Texas


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mosquito Aedes taeniorhynchus is an important epidemic vector of Venezuelan equine encephalitis virus (VEEV), but detailed studies of its infection are lacking. We compared infection by an epidemic VEEV strain to that by an enzootic strain using virus titrations, immunohistochemistry, and a virus expressing the green fluorescent protein. Ae. taeniorhynchus was more susceptible to the epidemic strain, which initially infected the posterior midgut and occasionally the anterior midgut and cardia. Once dissemination beyond the midgut occurred, virus was present in nearly all tissues. Transmission of the epidemic strain to mice was first detected 4 days after infection. In contrast, the enzootic strain did not efficiently infect midgut cells but replicated in muscles and nervous tissue on dissemination. Because VEEV emergence can depend on adaptation to epidemic vectors, these results show that epidemic/enzootic strain comparisons not only comprise a useful model system to study alphavirus transmission by mosquitoes, but also have important public health implications.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Understanding the infection of mosquitoes by arboviruses is necessary to elucidate the epidemiology of diseases caused by these agents. This topic has therefore received considerable attention in the past1–5 but only minor emphasis recently. Many barriers exist to the infection, dissemination, and transmission of arboviruses by their mosquito vectors, and an understanding of these mechanisms is important for the design of safer vaccines and novel strategies to interrupt transmission.

Most studies agree that posterior midgut epithelial cells of the mosquito are the primary sites of arbovirus replication after ingestion of a viremic bloodmeal.6–8 A threshold of infection, the minimum dose required to infect the midgut of 1–5% of mosquitoes, has been shown for many viruses.9 After infection and amplification of the virus in the midgut epithelium, escape from the midgut into the hemocoel (i.e., dissemination) where secondary tissues are infected including the salivary glands must occur for transmission to take place. Most arboviruses seem to disseminate through the hemolymph,5 although two studies suggest neural pathways.10,11 Many arboviruses are first detected in the salivary glands at the same time as other tissues in the hemocoel; therefore, it is not known whether amplification in tissues other than the midgut and salivary glands is needed for biologic transmission.5

An important emerging arbovirus lacking attention in recent years regarding virus/vector interactions is Venezuelan equine encephalitis virus (VEEV; Togaviridae: Alphavirus). The principal vector in most major coastal outbreaks, including the 1995 epidemic in Venezuela and Colombia involving ~100,000 people, is the mosquito Aedes (Ochlerotatus) taeniorhynchus.12,13 This species is more susceptible to most epidemic than to enzootic strains,14,15 and the adaptation of VEEV to this vector may be an important determinant of epidemic transmission and VEE emergence.16,17 However, no detailed studies of the infection, replication, and dissemination of VEEV in this vector have been reported. To more fully understand the differential susceptibility of Ae. taeniorhynchus, we compared the infection and dissemination patterns of an epidemic, subtype IC strain to that of an enzootic, subtype IE strain using virus titration, immunohistochemistry (IHC), and a virus expressing the green fluorescent protein (GFP). We also determined the earliest time-point when this mosquito can transmit to a vertebrate host.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Virus. VEEV strains were rescued from infectious cDNA clones derived from epidemic subtype IC strain 3908, enzootic subtype IE strain 68U201, or strain 3908 expressing GFP. Strain 3908 was isolated in 1995 from a febrile human during a major epidemic in Venezuela13 and was passaged once in C6/36 mosquito cells before undergoing infectious cDNA clone production.14 Enzootic strain 68U201 was isolated from a sentinel hamster in Guatemala in 1968 and was passaged once in suckling mice and twice in BHK cells before cDNA cloning.18 The GFP gene and an additional sub-genomic promoter were inserted between the structural and non-structural protein gene regions of the strain 3908 clone (3908/GFP). Virus recovered from BHK cells electroporated with transcribed RNA was used for all experiments without further passage. The use of virus derived from an infectious clone minimized attenuating mutations that occur when VEEV is passaged in cell culture.19

Mosquitoes. Aedes taeniorhynchus F1 mosquitoes were derived from adults collected in Galveston, TX (latitude, 29°13.13' N; longitude, 94°56.06' W). Mosquitoes were reared in an insectary at 27°C, 80% relative humidity, using a light/dark cycle of 12:12 hours. Larvae were fed a 1:1 mixture of TetraMin fish flakes (Doctors Foster and Smith, Thinelander, WI) and crushed Prolab 2500 rodent diet (PMI Nutrition International, Brentwood, MO). Adult females were infected 6–8 days after emergence by an artificial bloodmeal (see below) and incubated at 27°C with 10% sucrose provided ad libitum.

Mosquito infection for titration and immunohistochemistry. Mosquitoes were offered an artificial bloodmeal containing 20% (vol/vol) fetal bovine serum (FBS), 10% (vol/vol) Eagles minimal essential medium (MEM), and 70% (vol/vol) packed sheep red blood cells. Blood-meal titers were 5 (low titer) and 7 (high titer) log10 plaque forming units (PFU)/mL for VEEV strain 3908 and 7 log10 PFU/mL for strain 68U201; uninfected bloodmeals were used for controls. An additional cohort of mosquitoes was infected intrathoracically with ~1 µL containing 4 log10 PFU of each VEEV strain. Five mosquitoes per cohort were collected on days 1–11, 16, and 21 for trituration in 300 µL of 20% MEM using a Mixer Mill 300 (Retsch, Newton, PA), and titrated on Vero cells. Additionally, 3 mosquitoes/d were collected for fixation (10% formol saline) and paraffin embedding. To determine dissemination status, the legs and wings, which contain hemolymph, were removed for infectious virus assays, and mosquito bodies were injected intrathoracically with 10% formol saline. Mosquitoes were stored at 4°C in 1 mL of fixative for 24 hours and transferred to 1 mL of 70% ethanol until further processing. Legs/wings were triturated in 300 µL of 20% MEM, and 75 µL of the supernatant was added to Vero cells and observed for cytopathic effects (CPEs).

Mosquito transmission. Mosquitoes were infected with 7 log10 PFU/mL of VEEV strain 3908 in an artificial bloodmeal (described above). Cohorts of 10–24 fully engorged mosquitoes were sorted randomly into cohorts and, on selected days after infection (Table 1Go), were allowed to feed on a naïve mouse. Exposed mice were held in individual cages and monitored for signs of VEE. The animal studies were approved by the University of Texas Medical Branch Institutional Animal Care and Use Committee.


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TABLE 1
Transmission of VEEV strain 3908 to mice
 
Immunohistochemistry. VEEV antigen was detected by immunohistochemistry, which is an immunologically based method where an antibody raised against VEEV was used to localize the viral antigen. This primary antibody to VEEV was detected using a horseradish peroxidase–conjugated secondary antibody, which reacts with aminoethylcarbazole (AEC) peroxidase substrate, allowing for the visualization of antigen as a red precipitate. For this method, mosquitoes were embedded in paraffin,20 and 6-µm serial sagittal sections were dried on slides treated with Vectabond following the manufacturer’s protocol (Vector Laboratories, Burlingame, CA). Slides were incubated overnight at 56°C before deparaffinization in xylene and hydrated in a graded alcohol series. Slides were refixed in 10% neutral buffered formalin, and antigen was retrieved using proteinase K (Sigma-Aldrich, St. Louis, MO) prepared as a 50 mg/mL stock, diluted 1:500 in PBS. Endogenous peroxidase was inactivated in 3% hydrogen peroxide in methanol, and slides were blocked in 3% FBS in phosphate-buffered saline (PBS; blocking buffer). Primary antibody was VEEV mouse ascitic fluid (ATCC, Manassas, VA) diluted 1:300 in blocking buffer, and goat anti-mouse horseradish peroxidase–conjugated secondary antibody (KPL, Gaithersburg, MD) was diluted 1:1,000 in blocking buffer. Aminoethylcarbazole peroxidase substrate allowed the visualization of antigen as a red precipitate, and the slides were developed according to the manufacturer’s protocol (Enzo Life Sciences, Farmingdale, NY). Slides were counter-stained with Mayer’s hematoxylin (Poly Scientific, Bay Shore, NY) diluted 1:3 in water and mounted using aqueous medium (Daido Sangyo, Tokyo, Japan). Antigen staining was scored on a scale from 1 to 3: for light staining and very few cells of a specific tissue positive (1+), medium staining and approximately one half the cells positive (2+), and dark staining and more than one half the cells positive (3+).

Mosquito infection for fluorescence detection. In contrast to IHC, the use of a viral construct expressing GFP allowed for the direct visualization of virus through fluorescence microscopy without the need for immunologically based reagents. For this method, mosquitoes were infected with 7 log10 PFU/mL of VEEV strain 3908/GFP in an artificial bloodmeal or by intrathoracic inoculation as described above. Five mosquitoes per cohort were collected for titration on days 1–11, 16, and 21, 3 mosquitoes/d were collected for mid-gut and salivary gland dissection, and 3 mosquitoes/d were collected for frozen sectioning. Before dissection/sectioning, the legs/wings were removed for dissemination assays (see above). Dissected tissues were fixed in 4% paraformaldehyde (PFA) and mounted with ProLong Gold antifade reagent with 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR). Mosquitoes used for frozen sections were injected intrathoracically with 4% PFA, stored at 4°C in 1 mL of 4% PFA for 24 hours, transferred to 1 mL of PBS, and frozen in OCT compound (Sakura Finetek, Torrance, CA). Sagittal sections 6 µm were cut with a cryostat, and every fifth section mounted with DAPI. The dissected tissues and frozen sections were observed using an Olympus Fluo-View-1000 scanning confocal microscope (Olympus, Melville, NY) and scored for infection (described above).

Statistics. Mosquito titers were analyzed by two-way analysis of variance (ANOVA; virus x day effect) with day as the repeated measure, followed by Bonferroni post-tests for comparison between viruses on specific days. P < 0.05 was considered significant using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA) for statistical analyses.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mosquito titrations and transmission. Aedes taeniorhynchus mosquitoes infected orally exhibited considerable variability in mean virus titers (Figure 1Go). Many samples infected with VEEV strain 3908 at a low titer, strain 68U201, and strain 3908/GFP bloodmeals were below the assay’s limit of detection (0.4 log10 PFU); however, all mosquitoes infected with strain 3908 high titer bloodmeals had detectable levels of virus. A two-way ANOVA comparing the virus strain used and the day after infection revealed highly significant virus strain effects (P < 0.0001), day effects (P = 0.0036), and insignificant interactions (P = 0.0767). Because no significant interactions were detected, Bonferroni post-test P values for comparing viruses on specific days were not meaningful and are not shown.


Figure 1
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  		    FIGURE 1. Replication of VEEV in mosquitoes after oral infection. The titer of infection is followed by the VEEV strain used. N = 5 mosquitoes/d. Dotted line represents limit of detection, and values in parentheses below this line represent number of samples below this detection limit. For statistical analysis, these values were set in between zero and the limit of detection (0.2 log10 PFU). The lower error bars have been omitted for visual clarity. This figure appears in color at www.ajtmh.org.

 
In contrast with orally infected mosquitoes, very little variability in the mean virus titers was observed after intrathoracic infections (Figure 2Go). A two-way ANOVA comparing virus strain and day after infection revealed highly significant interactions (P < 0.0001) making P values for the virus strain effect (P < 0.0001) and day effect (P < 0.0001) difficult to interpret. According to the Bonferroni post-test, mosquitoes infected intrathoracically with strain 3908 contained significantly more virus than mosquitoes infected with strain 68U201 and 3908/GFP on Days 6, 7, 10, 16, and 21 and Days 1–3 and 6 after infection, respectively. Mosquitoes infected intrathoracically with 3908/GFP had significantly higher titers compared with 68U201-infected mosquitoes on Days 16 and 21 (Figure 2Go). However, because of the small sample size and natural variation, these statistical differences are most likely biologically meaningless because the general trends in virus titers within the intrathoracically infected mosquitoes seem uniform.


Figure 2
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  		    FIGURE 2. Replication of VEEV in mosquitoes after intrathoracic infection. The titer of the inoculum is followed by the VEEV strain used. N = 5 mosquitoes/d. Symbols represent P values from the Bonferroni post-tests where one symbol is considered significant, two symbols very significant, and three symbols extremely significant. *3908 vs 68U201; #3908 vs 3908/GFP; +68U201 vs 3908/GFP.

 
Exposure of mice to strain 3908–infected Ae. taeniorhynchus indicated that transmission can occur after as little as 4 days of extrinsic incubation (Table 1Go). In three separate experiments, initial transmission was detected on different days. Consistent transmission was not detected until Day 8 after infection.

IHC analysis of VEEV infection and dissemination. VEEV antigen was detected 24 hours after mosquitoes received the high titer strain 3908 bloodmeal. Within the posterior midgut, epithelial cells in the posterior portion were infected either singly or in clusters. Viral antigen was concentrated along the apical brush border and adjacent midgut lumen of epithelial cells, which was not seen in cellular detritus and was not apparent in negative controls (Figure 3Go). Additional infected tissues on Day 1 after infection included the anterior portion of the anterior midgut, the cardial epithelium, and the ventral and dorsal diverticula (Figure 4Go). By Day 2, viral antigen was detected in the intussuscepted foregut epithelial cells. In contrast, infected tissues were not observed in mosquitoes exposed to strain 68U201 and the low titer strain 3908 bloodmeals until Days 4 and 5, respectively. Then, only a few weakly staining epithelial cells were observed in the posterior portion of the posterior midgut. Within the posterior midgut, cell-to-cell spread or differential susceptibility of epithelial cells was suggested by the presence of adjacent, infected cells (Figure 3FGo).


Figure 3
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  		    FIGURE 3. Immunohistochemical staining of VEEV in the posterior midgut during early (A–D) and late (E–H) infection. A, Control mosquito, Day 1, x40. BB, brush border. B, High titer 3908–infected mosquito, Day 1, x40. C, High titer 3908–infected mosquito, Day 1, x20. Arrows point to infected posterior midgut epithelial cells (PMEs). D, High titer 3908–infected mosquito, Day 1, x10. Arrows point to antigen staining in the bloodmeal surrounding the posterior portion of the posterior midgut epithelium. E, Control mosquito, Day 8, x20. F, Low titer 3908–infected mosquito, Day 8, x20. G, High titer 3908–infected mosquito, Day 11, x40. H, High titer 3908–infected mosquito, Day 16, x40. CV, cellular vacuolization. This figure appears in color at www.ajtmh.org.

 

Figure 4
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  		    FIGURE 4. Immunohistochemical staining of VEEV in the anterior midgut (A–F) and hindgut (G–H). A, Control mosquito, Day 1, x10. B, High titer 3908–infected mosquito, Day 1, x10. CE, cardia epithelium. C, High titer 3908–infected mosquito, Day 1, x10. AME, anterior midgut epithelium. D, High titer 3908–infected mosquito, Day 1, x20. VDM, ventral diverticulum muscle. E, High titer 3908–infected mosquito, Day 7, x20. CoEl, corpus ellatum; Es, esophagus; IF, intussuscepted foregut. F, High titer 3908–infected mosquito, Day 11, x40. FB, fat body. G, Control, Day 10, x40. HGE, hindgut epithelium. H, 68U201-infected mosquito, Day 10, x40. This figure appears in color at www.ajtmh.org.

 
Dissemination from the mosquito midgut was determined either by the detection of infectious virus in the mosquito legs and wings or by antigen detection outside of the midgut (comprising the posterior and anterior midgut, and cardia) in the hemocoel associated cells and tissues. Dissemination (infectious virus) was initially detected in mosquitoes infected with the high titer strain 3908 bloodmeals by Day 2 (Figure 5Go), and by Day 4 by antigen detection in hemocoel-associated tissues such as the intussuscepted foregut, hindgut, abdominal, thoracic, and cephalic ganglia (Figures 4Go, 6Go, and 7Go), and the abdominal and thoracic fat body. Other tissues infected on Day 4, but that are not indicative of dissemination, included the cardia and posterior midgut. The salivary glands were not found to be infected until Day 7 (Figure 6B–DGo), and the anterior portions of the lateral lobes were most often antigen positive. By Day 11, viral antigen was abundant throughout the alimentary tract (Figures 3GGo, 4FGo, and 6DGo), as well as in the fat body (Figure 8BGo) by Day 16. Other tissues infected on Day 16 included the Malpighian tubules and ovarian follicles (Figure 8C, D, FGo). The only sign of pathology, observed on Day 16 and only in a single mosquito, was cellular vacuolization in the posterior midgut (Figure 4HGo), which was not observed in un-infected controls.


Figure 5
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  		    FIGURE 5. Dissemination of VEEV to the legs and wings of orally infected mosquitoes. The titer of infection is followed by the VEEV strain used.

 

Figure 6
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  		    FIGURE 6. Immunohistochemical staining of VEEV in the salivary glands (A–D) and thoracic ganglia (E and F). A, Control mosquito, Day 8, x10. SG, salivary gland; TG, thoracic ganglia. B, High titer 3908–infected mosquito, Day 7, x20. C, High titer 3908–infected mosquito, Day 8, x40. D, High titer 3908–infected mosquito, Day 11, x10. Es, esophagus; CE, cardia epithelium; IF, intussuscepted foregut; VDM, ventral diverticulum muscle. E, High titer 3908–infected mosquito, Day 4, x20. CB, cell body. F, 68U201-infected mosquito, Day 6, x40. GC, ganglionic connective tissue. This figure appears in color at www.ajtmh.org.

 

Figure 7
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  		    FIGURE 7. Immunohistochemical staining of VEEV in the nervous tissue (A–F) and sense organs (G–H). A, Control mosquito, Day 7, x20. AG, abdominal ganglia. B, High titer 3908–infected mosquito, Day 4, x20. CB, cell body. C, High titer 3908–infected mosquito, Day 8, x20. CB, cell body. D, Control mosquito, Day 8, x20. CG, cephalic ganglia; Om, ommatidia; JO, Johnston’s organ. E, High titer 3908–infected mosquito, Day 7, x40. F, High titer 3908–infected mosquito, Day 8, x20. GC, ganglionic connective tissue G, 68U201-infected mosquito, Day 5, x20. H, 68U201-infected mosquito, Day 6, x40. This figure appears in color at www.ajtmh.org.

 

Figure 8
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  		    FIGURE 8. Immunohistochemical staining of VEEV in the excretory (A–D) and reproductive (E and F) systems. A, Control mosquito, Day 7, x40. FB, fat body. B, High titer 3908–infected mosquito, Day 16, x20. C, High titer 3908–infected mosquito, Day 16, x40. MT, Malpighian tubule. D, High titer 3908–infected mosquito, Day 16, x40. E, Control mosquito, Day 5, x40. C, calyx; OF, ovarian follicle. F, High titer 3908–infected mosquito, Day 16, x40. This figure appears in color at www.ajtmh.org.

 
Dissemination (infectious virus detection) from the midgut of mosquitoes orally infected with the low titer strain 3908 bloodmeal was detected by Day 5 (Figure 5Go) and by Day 7 by the presence of antigen in the intussuscepted foregut and the thoracic fat body. The only other tissue found to be infected on Day 7, but which is not indicative of dissemination, was the cardia. By Day 8, the anterior portion of the anterior midgut was infected. Not until Day 21 was more widespread dissemination noted in low-titer strain 3908 infections, in the abdominal and cephalic ganglia, and in the abdominal, thoracic, and cephalic fat body.

Dissemination from the midgut of mosquitoes orally infected with strain 68U201 was detected by Day 4 in the legs/wings (infectious virus; Figure 5Go). The only tissue outside of the posterior midgut with detectable antigen on Day 4 was the cephalic ganglion (Figure 7GGo). By Day 6, the intussuscepted foregut, abdominal, thoracic, and cephalic ganglia, fat body, and Johnston organ all contained VEEV antigen. By Days 9 and 10, muscles of the gut, but not epithelial cells, were also infected.

GFP-labeled VEEV dissemination. The VEEV 3908/GFP virus exhibited reduced mosquito replication in comparison with its wild-type counterpart containing no reporter gene (Figures 1Go and 2Go). Mosquitoes infected orally did not develop a disseminated infection until Day 4 (Figure 5Go). In contrast, GFP fluorescence was detected weakly by Day 1 in mosquitoes infected intrathoracically, and by Day 4 GFP was found in most tissues such as the posterior midgut, fat body, intussuscepted foregut, esophagus, cardia, anterior midgut, rectal area, the anterior portion of the salivary gland lateral lobes, the cephalic ganglia, and the abdominal and thoracic ganglia (Figure 9A–HGo). By Day 8, epithelial cells of the anterior and posterior midgut (Figure 9I–JGo) expressed GFP, along with cells in the ovary calyx and Malpighian tubules (Figure 9K–LGo).


Figure 9
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  		    FIGURE 9. Confocal micrographs of mosquitoes infected intrathoracically with VEEV strain 3908 expressing GFP. The transmitted image is overlayed with DAPI and GFP images. A–H, Day 4 after infection. I–L, Day 8 after infection. A, PME, posterior midgut epithelium; CM, circular muscle. B, FB, fat body. C, Es, esophagus; IF, intussuscepted foregut; CE, cardia epithelium; CM, circular muscle; LM, longitudinal muscle. D, Rectal area. E, SG, salivary gland. F, CG, cephalic ganglion; CB, cell body; GC, ganglionic connective tissue; N, neuropile. G, AG, abdominal ganglion. H, TG, thoracic ganglion. I, AME, anterior midgut epithelium. J, PME, posterior midgut epithelium. K, Ovary; C, calyx. L, MT, malpighian tubule. Red bar in lower corner represents 20 µm. This figure appears in color at www.ajtmh.org.

 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Understanding the movement of arboviruses from the lumen of the mosquito midgut into the hemocoel, which is required for virus transmission by mosquitoes, is important for understanding vector–virus interactions. This study contributes to our understanding of the initial infection, dissemination, and transmission of two VEEV subtypes in the epidemic mosquito vector, Ae. taeniorhynchus.

Mosquito titrations. A high level of variability in mean virus titers was observed after mosquitoes were infected orally (Figure 1Go), in contrast to little variability after intrathoracic injection (Figure 2Go). This finding underscores the sporadic nature of VEEV oral infection and dissemination from the midgut, most likely because of midgut infection and/or escape barriers. Although significant differences in virus titers were found on particular days after infection for mosquitoes infected intrathoracically, the overall trend in the virus titer was similar for both the epidemic and enzootic virus strains.

IHC analysis of VEEV infection and dissemination. In agreement with other studies of alphaviruses,6–8 the posterior midgut epithelial cells were the initial site of VEEV replication, which was detected within 24 hours of a high titer strain 3908 bloodmeal. In addition to the cytoplasm of midgut epithelial cells, antigen staining appeared concentrated along the brush border and in the surrounding lumen, which was not observed in negative controls or cellular detritus (Figure 3Go). Further studies are needed to confirm that viral antigen accumulates along the brush border and to determine whether VEEV is shed into the lumen. Additional, initial sites of replication in Ae. taeniorhynchus infected with high titer strain 3908 bloodmeals included epithelial cells of the anterior portion of the anterior midgut and the cardia (Figure 4Go). Dissemination was detected by Day 2, when the intussuscepted foregut contained viral antigen. It is possible that VEEV spreads in a cell-to-cell manner from the cardia to the adjacent intussuscepted foregut, where it could hypothetically escape into the hemocoel without traversing a basal lamina.21 Because a disseminated infection occurred in some mosquitoes even before infection of the intussuscepted foregut was detected, virus in these cases probably disseminated into the hemocoel through the posterior midgut. A similar pattern of infection was observed for mosquitoes infected with the low titer strain 3908 bloodmeal, although dissemination occurred much later (Day 7) compared with mosquitoes infected with the higher dose.

Several other arboviruses, including two alphaviruses, have been found to replicate in the anterior region of the midgut soon after infection.16,21–25 Studies of Western equine encephalitis virus using hanging drop bloodmeals suggest that anterior midgut infection could be an artifact of artificial bloodmeals.26 We found that mosquitoes that feed from hanging drops accumulate more blood in their diverticulum compared with those that feed through an artificial membrane (DRS, unpublished data). This could lead to infection of the anterior midgut when the bloodmeal is gradually directed from the diverticulum to the midgut. This difference in the bloodmeal being deposited in the diverticulum compared with the midgut may be caused by the penetration of the membrane by the mosquito’s proboscis and deserves further study. We did observe antigen staining in the diverticulum on Day 1 after infection with the high titer strain 3908 bloodmeal, suggesting a disseminated infection. However, no infectious virus was detected in the legs and wings of the mosquito; therefore, this could represent non-specific staining because VEEV presumably cannot infect this organ directly from the diverticular lumen caused by the impervious, luminal cuticular lining.25,26 Electron microscopy is needed to clarify this observation.

In contrast to mosquitoes infected with a high titer strain 3908 bloodmeal, strain 68U201 and low dose strain 3908 bloodmeals did not result in detectable antigen in the posterior midgut until Days 4 and 5, respectively. The virus was likely undetectable before Day 4 because of a low level of replication, below the detection limit for our assays (~3 log10 PFU/g of tissue). Once VEEV escaped into the hemocoel, amplification mainly occurred in epithelial cells of the gut, neural tissue, and fat body for mosquitoes infected with strain 3908. In contrast, when strain 68U201 escaped into the hemocoel, amplification primarily occurred in muscles surrounding the gut (not epithelial cells) and neural tissues. This may reflect a dearth of strain 68U201–specific receptors on the epithelial cells.

For both VEEV strains, the nervous tissues of mosquitoes were frequently infected. Several studies with dengue viruses report heavy infection of the mosquito nervous system.27–29 Platt and others30 showed that Ae. aegypti infected with dengue-3 virus need more time to feed on a vertebrate than un-infected mosquitoes, which could enhance transmission. Because ours and previous studies31,32 showed VEEV infection of mosquito nervous tissue, it would be interesting to determine if infection alters Ae. taeniorhynchus behavior.

Salivary gland infection is needed for horizontal, biologic transmission of arboviruses. Mosquitoes infected with the high titer strain 3908 bloodmeal first had detectable viral antigen in the salivary glands by Day 7, beginning with the anterior portion of the lateral lobe. Weaver32 reported that the salivary glands of Culex (Melanoconion) taeniopus mosquitoes are first infected with VEEV by Day 4, consistent with initial transmission on Day 5.33 Our results are similar, in that the earliest VEEV transmission was detected on Day 4, although high variability continued until Day 8 (Table 1Go). Strain 68U201, and strain 3908 after a low dose, were not detected in the salivary glands, possibly because of a salivary gland infection barrier and/or to limited amplification in the hemocoel.

Additional organs infected by the high titer strain 3908 bloodmeals included the Malpighian tubules and ovaries. These findings contrast with those of Weaver,32 who reported no VEEV strain 68U201 in these organs of the enzootic vector, Cx. taeniopus. Larsen and Ashley31 did report VEEV subtype IAB in the ovaries and Malpighian tubules of Ae. aegypti, but did not detect virus by electron microscopy in the ovarian follicles. These differences are most likely caused by different virus strain/mosquito species interactions.

We detected signs of pathology (cellular vacuolization) only late in infection in the posterior midgut of a single mosquito infected with a high oral dose of strain 3908 (Figure 3HGo). Two other alphaviruses cause cytopathology in their mosquito vectors; Western equine encephalitis virus (WEEV) causes vacuolization and luminal extensions of midgut epithelial cells of Culex tarsalis, and Eastern equine encephalitis virus (EEEV) causes ultrastructural changes in the posterior midgut epithelial cells of Culiseta melanura.34,35

Analysis of GFP-labeled VEEV dissemination. The VEEV strain 3908/GFP exhibited reduced replication in comparison to wild-type VEEV with no reporter gene (Figures 1Go and 2Go). Orally infected 3908/GFP mosquitoes did not develop a disseminated infection until much later than mosquitoes infected with wild-type strain 3908 (Figure 5Go). In contrast, GFP was detected weakly by Day 1 after infection and by Day 4 was seen throughout most tissues in intrathoracically infected mosquitoes (Figure 9Go). Detection of strain 3908 replication using GFP or IHC generally yielded consistent results. One exception was GFP detection in the ovary calyx, whereas IHC viral antigen was detected only in a few ovarian follicles.

In conclusion, our results underscore the importance of the midgut barriers to infection and dissemination in natural VEEV–vector interactions. The first day VEEV epidemic strain 3908–infected mosquitoes can transmit to a vertebrate host is Day 4 after infection, although this timing is highly variable. After the replication of strain 3908 in the epithelial cells of the posterior midgut and sometimes in the anterior midgut and cardia, virus disseminates and infects additional epithelial cells, muscles, and nervous tissue before infection of the salivary glands. In contrast, enzootic VEEV strain 68U201 does not efficiently infect midgut epithelial cells and, when dissemination beyond the midgut occurs, the muscles associated with the gut and nervous tissue serves as important replication sites. Because VEE emergence can depend on adaptation to epidemic vectors, these differences in infection, dissemination, and transmission not only make up a useful, comparative model system to study alphavirus transmission by mosquitoes, but also have important public health implications.

Received December 27, 2006. Accepted for publication April 10, 2007.

Acknowledgments: The authors thank Jing Huang for rearing mosquitoes and Nikolaos Vasilakis and Slobodan Paessler for technical advice. We also thank Charles Fulhorst, Lifang Zhang, and James Grady for statistical advice and William Romoser for kindly reviewing this manuscript. DRS was supported by a fellowship from the Keck Virus Imaging Program and by CDC Training Grant T01/CCT622892, and this research was supported by NIH Grants AI418807 and AI57156.

* Address correspondence to Darci R. Smith, Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609. E-mail: darci.smith{at}amedd.army.mil Back

Authors’ addresses: Darci R. Smith, Nicole C. Arrigo, Grace Leal, 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. Linda E. Muehlberger, Histopathology Service Core, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1019, Telephone: 409-747-0735, Fax: 409-747-0725.

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


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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