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    Spatial separation of donor and recipient mosquitoes feeding on a mouse.

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    Quantitative reverse transcription-polymerase chain reaction of West Nile Virus (WNV)-infected salivary glands dissected from intra-thoracically infected Culex pipiens quinquefasciatus mosquitoes over a 21-day time course of infection (•). PFU = plaque-forming units. Error bars represent standard deviation.

  • View in gallery

    A, West Nile virus within a Culex, p. quinquefasciatus saliva cavity, 28 days post-infection (d.p.i.). Virus particles (Vp) line the saliva (Sa) cavity along the epithelial cell membrane and surround an unknown salivary component (USC). Cy = cytoplasm; Mi = mitochondrion; Va = vacuole. B, negative stain of mosquito saliva 15 d.p.i. Inset, high-power magnification of an individual virus aggregate. Arrows = single virus particles; arrowheads = virus aggregates; pSa = grainy, dark-staining areas putatively identified as mosquito saliva.

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NONVIREMIC TRANSMISSION OF WEST NILE VIRUS: EVALUATION OF THE EFFECTS OF SPACE, TIME, AND MOSQUITO SPECIES

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  • 1 Department of Pathology, University of Texas Medical Branch, Galveston, Texas

To evaluate the potential for nonviremic transmission (NVT) of West Nile virus (WNV) to occur in nature, we examined the effect of increasing spatial and temporal separation between co-feeding mosquitoes on the efficiency of nonviremic transmission and the potential of a West Nile virus bridge vector species, Aedes albopictus, to be infected via nonviremic transmission. West Nile virus-infected (donor) Culex pipiens quinquefasciatus were allowed to feed on a mouse for 5 minutes followed by non-infected (recipient) mosquitoes with increasing spatial (0, 10, 20, 30, 40, or 50 mm) or temporal (0, 15, 30, 45, or 60 min) separation from the site or time of donor feeding, respectively. Recipients became infected when feeding up to 40 mm from the donor and up to 45 minutes after donor feeding. Additionally, nonviremic transmission of West Nile virus from Cx. p. quinquefasciatus to Ae. albopictus was observed.

INTRODUCTION

The 1999 introduction of West Nile virus (WNV), family Flaviviridae genus Flavivirus, an arthropod-borne virus (arbovirus) into North America resulted in widespread dispersal of the virus and the establishment of seasonal activity patterns associated with severe disease and human fatalities (www.cdc.gov/ncidod/dvbid/westnile). The natural cycle of WNV typically involves ornithophilic Culex mosquitoes feeding on avian hosts,1 although WNV has been detected in at least 75 different arthropod species2,3 including at least 60 North American mosquito species (http://www.cdc.gov/ncidod/dvbid/westnile/mosquitoSpecies.htm). In Texas, Culex pipiens quinquefasciatus has been identified as the primary vector of WNV4 whereas other Culex spp. mosquitoes, such as Cx. p. pipiens and Cx. tarsalis are more important in other regions. There have been numerous laboratory studies on the competence of various N. American mosquitoes to become infected with and subsequently transmit WNV.512 The unprecedented rate at which WNV disseminated in North/Central America suggests that multiple transmission mechanisms may contribute to the life cycle; indeed routes of infection that are unusual for an arbovirus have been documented.13 Recently nonviremic transmission (NVT) of WNV from infected to uninfected Cx. p. quinquefasciatus mosquitoes was demonstrated and implicated as a potential contributor to the rapid dispersal of WNV.1416

Nonviremic transmission is defined as virus transmission between two arthropods feeding on the same vertebrate host before the development of a classic viremia, subsequent to replication in target tissue. NVT results in rapid and direct transmission from infected donor to uninfected recipient arthropods thus eliminating the need for an intrinsic incubation period (development of viremia subsequent to replication), and potentially increasing the range of vertebrate hosts capable of facilitating mosquito to mosquito transmission.15 This phenomenon has been well documented with respect to tick-borne pathogens such as Thogoto virus (THOV), Louping ill virus (LIV), Tick-borne encephalitis virus (TBEV), and Borrelia burgdorferi.1723 Nonviremic transmission of WNV by rapidly feeding Argasid ticks has also been described.24 Additionally, NVT of Vesicular stomatitis virus has been observed to occur between black flies co-feeding on deer mice.25 NVT of TBEV is a well-characterized phenomenon that has been demonstrated to occur between arthropods feeding on immune natural rodent hosts22 and has been identified as a critical mechanism for the maintenance of TBEV in nature.26

It has been hypothesized that NVT is likely to be most efficient when both infected and uninfected arthropods feed in close spatial and temporal proximity.14,27 The rationale is that with increasing time and distance, infectious virus becomes less accessible for infection of recipients due to dilution or diffusion from the feeding site, sequestration by immune cells, neutralization by antibody, or some other mechanism resulting in reduced availability/infectivity. Although this assumption is logical there are relatively few experimental observations to support it. In experiments with Ixodes spp., donor and recipient feeding times have invariably overlapped. In the studies of TBEV NVT it was concluded that spatial separation of donor and recipient ticks had a greater impact on NVT efficiency than duration of co-feeding,22 whereas Jones and Nuttall reported significant decreases in THOV transmission efficiency resulting from decreasing temporal tick feeding association.28 Here we report the effects of incrementally increasing either the spatial or temporal separation between co-feeding mosquitoes and the potential for NVT to facilitate infection of Aedes albopictus (a potential WNV bridge vector species).8

MATERIALS AND METHODS

Mosquitoes.

Cx. p. quinquefaciatus mosquitoes were laboratory-reared from a colony strain (Sebring Strain) originally collected from Sebring County, Florida in 1998. Colony mosquitoes used for these experiments were > F30 generation. The Sebring strain of Cx. p. quinquefasciatus has been well characterized with respect to WNV susceptibility.15,2931 Ae. albopictus mosquitoes were field collected in Galveston, Texas in summer 2003; mosquitoes used for theses experiments were F2 generation. Mosquitoes were maintained at 28°C on a 14:10 hour light:dark cycle including a 1-hour period to simulate dusk/dawn conditions. Larvae were fed using a 1:1 mixture of TetraMin fish flakes and ground Prolab 2500 rodent diet and adults were allowed to feed freely on a 10% sucrose solution. Additionally adult females were blood-fed once weekly on anesthetized hamsters in accordance with National Institutes of Health and UTMB humane laboratory animal use standards.

Virus.

West Nile virus stock for these experiments was derived from a single passage in Vero cells (green monkey kidney cells) of a 2002 Houston isolate (lineage 1) obtained as a brain/liver homogenate from an infected blue jay (Cyanocitta cristata), designated strain 114 (GenBank accession number AY187013).32 Stock titer was 2 × 108 plaque forming units (pfu)/mL, and sequence analysis confirmed homology with the 1999 New York strain (NY99 GenBank accession number AF196835).

Mosquito infection.

Donor infection.

Donor Cx. p. quin-quefasciatus mosquitoes were chilled for 60 seconds in a −20°C freezer and subsequently kept immobilized in a cold glass Petri dish on ice. WNV stock (0.5 μL) (titer 2 × 108 pfu/ml) was diluted 1:3 in Liebovitz L-15 media supplemented with 10% fetal bovine serum, 1% penicillin streptomycin, and 1% glutamine (Mediatech, Inc. cellgro®, Herndon, VA) and directly inoculated into mosquitoes intra-thoracically (IT). IT inoculations were preformed on a chill table, visualized through a dissecting microscope, and delivered using a manual pressure injection system. Virus (0.5 μL) was inoculated via the use of a calibrated glass needle. After injections mosquitoes were transferred to 1 pint cardboard cages with mesh lids and housed in a sealed humidified container, provided with 10% sucrose solution, and maintained in a Precision model 818 environmental chamber (Precision, Winches-ter, VA) at 28°C, with a 14:10 hour light:dark cycle.

Infectious feeds.

All infectious feeds were performed in an isolated glove box housed in a Biosafety Level 3 facility in accordance with National Institutes of Health and UTMB humane laboratory animal use standards. At all times donor and recipient mosquitoes were confined to separate sealed feeding chambers to preclude the possibility of mixing.

Nonviremic temporal feeds.

At 7 to 14 days post inoculation (d.p.i.), donor mosquitoes were transferred to a clean clear plastic cage with a fine mesh top. A shaved anesthetized ≥ 8-week-old outbred Swiss Webster mouse (Harlan Indianapolis, Indiana) was placed on top of the cage and mosquitoes were observed for the commencement of probing/ feeding. After donors probed/fed for 5 minutes the mouse was removed and maintained under anesthesia for the desired time period (15, 30, 45, or 60 min). After the appropriate time had elapsed, 100 recipient mosquitoes were allowed access to the mouse for 1 hour.

Nonviremic spatial feeds.

Cx. p. quinquefasciatus donor mosquitoes ≤ 3 were allowed to probe/feed at a defined 10 mm diameter exposure zone on an anesthetized mouse. Donor feeding was stopped after 5 minutes had elapsed and 100 recipients were immediately allowed to feed for 1 hour at a defined 25 mm diameter exposure zone (Figure 1). Donor and recipient exposure zones were separated from each other by zero (donors/recipients fed at the same 25 mm exposure zone), 10, 20, 30, 40, or 50 mm.

Nonviremic transmission with a bridge-vector.

Feeds using Ae. albopictus as recipients were conducted by allowing recipients to feed from the same exposure zone immediately after donor Cx. p. quinquefasciatus feeding.

For all experiments, donor mosquitoes were frozen at −80°C until titration for quantity of WNV. Blood was collected from the anesthetized mouse via cardiac stick or retro-orbital bleed immediately after the completion of the 1-hour donor feeding period. Serum was separated and frozen at −80°C until titration for quantity of WNV. Recipient mosquitoes were sorted and engorged females were incubated for 14 days at 28°C. Recipients that survived to day 14 post–feed were frozen at −80°C for subsequent analysis.

Mosquito and serum analysis by titration and real time reverse transcription-polymerase chain reaction.

West Nile virus donor mosquitoes, recipient mosquitoes, and serum samples were analyzed for virus by titration. Individual mosquitoes were triturated in a 2.0 mL round bottom safe-lock microcentrifuge tube (Eppendorf) containing 1 mL Liebovitz L-15 medium supplemented as previously described (with the addition of 500 μg/mL Amphotericin-B [Sigma Aldrich]) and a single zinc coated steel grinding ball (Daisy® Rogers, Arkansas) using a Retsch Mixer Mill MM301 operating at a frequency of 26 vibrations/sec for 4 minutes, followed by centrifugation at 13,000 × g for 6 minutes. To identify infected recipient mosquitoes, uniquely numbered triturates were loaded in duplicate 100 μL per well in a 96 well plate (Nunclon™, Roskilde, Denmark) and titrated for virus (100 and 10−1) on Vero cells. Titration plates were incubated for 7 days at 37°C and examined daily for cytopathic effect (cpe). Recipient mosquitoes identified as virus-positive were also tested using a WNV reverse transcriptase real time polymerase chain reaction assay as previously described.31 WNV donor mosquitoes, recipient mosquitoes identified as positive, and serum samples were analyzed by virus titration.33 Briefly, 10-fold serial dilutions were performed in duplicate in 96 well plates and seeded with Vero cells. Titers were calculated as tissue culture infectious dose 50 per mL (log10 TCID50/mL).34

Quantification of virus in salivary glands of intra-thoracically infected mosquitoes.

Salivary glands were dissected from WNV infected mosquitoes every other day over a 21-day time course post IT inoculation and placed in 10 μL 0.15 M NaCl. Salivary glands were lysed by addition of 10 μL of 0.1% Trition X-100, then vortexed for 60 seconds and centrifuged at 10,000 × g for 60 seconds.35 Supernatant (10 μL per salivary gland) was extracted and WNV quantified as previously described.31,36

Transmission electron microscopy (TEM).

Salivary glands were dissected from WNV-infected mosquitoes 28 days post-infection and processed for TEM analysis as previously described.29 For negative staining of saliva, mosquitoes were injected with 1 μ L of 9 log10 TCID50/mL of WNV and maintained for 15 days prior to saliva collection. For saliva collection, mosquitoes were aspirated from cartons and chilled on ice, at which point wings and legs were removed. Using sharp dissecting needles, the mosquito stylets were released from their outer sheath, or prementum. Mosquitoes were then placed on their sides and 2–3 μL of pilocarpine was brushed onto the thorax to induce salivation. The stylets were then inserted into a drop of sterile-filtered water suspended on a Formvar-carbon coated grid and allowed to salivate into water for 5 to 10 minutes. Water was allowed to dry (almost completely) at room temperature after which grids were treated with TEM fixative30 for 10 minutes, also at room temperature. Fixative was removed and grids were placed on several drops of water, then on a drop of 2% aqueous uranyl acetate for 30 seconds. The grids were analyzed immediately in a Philips CM100 or Philips 201 electron microscope at 60 kV.

RESULTS

Intra-thoracic inoculation of Cx. p. quinquefasciatus donor mosquitoes with 0.5 μL of a 1:3 dilution of WNV stock 2 × 108 pfu/mL consistently resulted in 100% of mosquitoes having a disseminated infection at 7 d.p.i (Figure 2). Donors were used for experimental feeds between 7 and 14 d.p.i. at which time individual salivary gland WNV titers did not significantly vary (Figure 2) and were known to facilitate transmission. IT-inoculated donor titers were 6.61 ± 0.57 and 7.66 ± 0.40 log10 TCID50/mL for temporal and spatial experimental feeds respectively, and 7.61 ± 0.19 log10 TCID50/mL in the experiments using Ae. albopictus as the recipient species. Previous data indicated that a single donor mosquito could efficiently support NVT of WNV.15 Because we have often observed that mosquitoes housed individually are reluctant to feed we used one to three donor mosquitoes per carton. This protocol frequently resulted in at least one mosquito feeding and most closely mimicked natural transmission in which relatively low numbers of WNV-infected mosquitoes and high numbers of non-infected mosquitoes would be expected.

Recipient mosquito feeding sites or times were incrementally increased with respect to proximity to donor feeding by restricting either the skin site or time of exposure, respectively. When recipient mosquitoes were allowed to feed at the donor exposure zone immediately after donor feeding, NVT was observed in 60% (3/5) of the replicate experiments with 1.52% and 6.14 ± 0.33 log10 TCID50/mL average efficiency and titer respectively (Table 1). In temporal experiments donor mosquitoes were allowed to probe and/or feed along the length of the ventral surface of an anesthetized mouse. Subsequent to 5 minutes of feeding the mouse was removed and maintained under anesthesia until a predetermined time period (15, 30, 45, or 60 min) had elapsed. Recipient mosquitoes were then permitted to feed on the ventral surface of the mouse for 1 hour. NVT was detected when “co-feeding” was separated by 15 and 45 minutes at efficiencies of 2.94% and 1.19%, respectively (Table 2).

The effects of increasing the distance between donor and recipient feeding sites were evaluated by restricting feeding using impermeable barriers that only permitted mosquito feeding at defined zones on a mouse. NVT was detected when co-feeding was spatially separated by up to 40 mm (Table 1). The highest rate of NVT (12.24%) was detected when co-feeding was spatially separated by 20 mm and was concurrent with a serum titer of 2.95 log10 TCID50/mL. When Ae. albopictus mosquitoes were allowed to feed at the donor exposure zone immediately after donor feeding, a single positive recipient (7.52 log10 TCID50/mL) was detected in one replicate (Table 1).

DISCUSSION

Lord and Tabachnick (2002) reviewed viremic and non-viremic transmission of arboviruses and formulated a general model of arbovirus transmission. A fundamental assumption in this model was that NVT “will depend on an infected and uninfected vector feeding sufficiently close together in time and space.”14,27 Jones and Nuttall manipulated the duration of co-feeding ticks, and reported that whilst 80% of recipients became infected with THOV if they co-fed with donors for 5 days, none became infected if co-feeding was limited to 3 days, and observed that recipient ticks became infected when fed 160 mm apart.28 The effects of space and time on NVT of TBEV by Ixodes ricinus ticks have been investigated by housing ticks in two separate chambers, which were placed next to each other on rodent hosts.22 The first contained both donor and recipient ticks; the second chamber contained only recipients. NVT of TBEV was observed in up to 44% of recipients co-feeding with infected donors for only a single day. Under all experimental conditions, infection of recipients was consistently higher when housed in the same container as the donors, compared with when they were housed separately.

The rapidity of mosquito-borne NVT precludes the possibility of virus replication in the vertebrate host during donor and recipient feeding. One possible explanation for mosquito-borne NVT is that the donor inoculates virus directly into the circulatory system at a dose that is below the limit of conventional detection methods,37 or that infectious virions are rapidly removed from circulation so that the probability of detection is low. Transmission in this way would not be driven by a classic replication-dependent viremia but instead would result from virus that is being directly delivered into the blood stream and imbibed by naïve mosquitoes without replication in the vertebrate host. Another possible mechanism for NVT is that virus is transferred from donor to recipient via the skin. Demonstrations that virus transmission from donor to recipient ticks was correlated with infection in the skin at the sites of tick feeding and not with viremia or generalized skin infection, suggest that virus was being preferentially recruited to sites of tick feeding, leading authors to hypothesize that viremia is a product of NVT rather than a prerequisite for transmission.20 However, because the temporal and spatial characteristics and the mechanics of tick and mosquito feeding are quite different, caution must be taken when extrapolating conclusions between these two systems.

Previously we reported NVT of WNV between co-feeding Cx. p. quinquefasciatus mosquitoes—a phenomenon not previously described for mosquito-borne viruses.15 Co-feeding experiments using single donors produced a 2.3% NVT rate, 7 donors a 2.0% NVT rate, and 57 donors a 2.4% NVT rate. In these experiments an anesthetized mouse was placed on a carton containing WNV-infected mosquitoes, which were allowed to probe and feed for approximately 5 minutes. After this initial exposure to infected mosquitoes the mouse was repositioned to allow simultaneous co-feeding of WNV infected and uninfected mosquitoes for 1 hour. Due to the unrestricted nature of this feeding protocol and the repositioning of the mouse we can envision at least three potential feed interactions: (1) donor and recipient mosquitoes feeding in close spatial and temporal proximity, (2) recipient mosquitoes feeding at skin sites previously penetrated or probed by donors, and/or (3) donors and recipients feeding separated either spatially or temporally. To elucidate the conditions required to facilitate NVT of WNV we incrementally increased either the temporal or spatial proximity between co-feeding mosquitoes to evaluate its effect on NVT efficiency.

We observed transmission of WNV between co-feeding mosquitoes that were separated by up to 40 mm, demonstrating that NVT is capable of facilitating infection of naïve mosquitoes feeding at distal sites. This suggests a systemic mechanism and circulatory involvement. In a single feed at 20 mm, 12.24% (6/49) of recipients became infected and subsequently developed relatively high titers of WNV (5.18 ± 0.22 log10 TCID50/mL). Interestingly 2.95 log10 TCID50/mL WNV was detected in the serum harvested from the mouse used for this feed. Because blood was collected immediately after recipient feeding (~1 hour after the completion of donor engorgement), we must conclude that virus detected in the serum was delivered to the blood by donor feeding and was not a product of replication in the mouse. Additionally, NVT of WNV was observed to occur when a 45-minute interruption between donor and recipient feeding was imposed, indicating that infectious virus remains available for a relatively long period after delivery by the donor. Western equine encephalitis virus artificial viremias lasting up to 90 minutes post needle inoculation demonstrate the relatively long systemic availability of arboviruses in vivo for arthropod vectors subsequent to virus injection.38,39 Further experiments are necessary to determine the temporal availability of various known doses of virus inoculated intravenously to assess the “infectious period” subsequent to an infected mosquito bite. These observations indicate that previous assumptions about the restrictions on vector co-feeding required to facilitate NVT of arboviruses may not be as stringent as initially hypothesized, and as such require reevaluation. Therefore a careful distinction must be made to distinguish tick-borne NVT, which requires the propagation of virus in lymphocytes, and trafficking via the lymphatics, from mosquito-borne NVT, which in the case of WNV can be systemic transmission via a non-propagative or non-replicative viremia-driven mechanism. Although these observations demonstrate that WNV can be systemically transmitted up to at least 45 minutes after donor feeding, they do not preclude the possibility of localized transmission due to feeding/probing at contaminated skin sites.

Under the experimental conditions used in these studies, the rate of NVT did not correlate directly with the temporal and spatial separation of donor and recipient feeding. For example NVT of WNV occurred at 20 and 40 mm but not at 30 mm, and occurred at 15 and 45 minutes but not at 30 minutes. Additionally transmission events for any one set of experimental condition were relatively infrequent, not occurring in all feed replicates. Traditionally vector competence is measured as a function of a threshold oral infectious dose and the relative contribution of vertebrate hosts assessed based on their ability to support sufficient viremia. Comparison of this traditional model of arbovirus transmission with a probabilistic model led to the conclusion that at any given concentration of virus there is a probability that a mosquito will become infected after bloodmeal ingestion.40 In previous NVT experiments it was noted that there was no direct correlation between the number of donors and the NVT rate, with no significant difference in transmission efficiency resulting when a range of 1 to 57 donors was used.15 In nature vertebrates are likely to be fed upon by relatively few infected mosquitoes at a given time. Because if NVT occurs in nature, then it is probably a consequence of a single infected bite, we designed our experiments to simulate these conditions. Analysis of the concentration of WNV salivated by individual mosquitoes has identified considerable variability in the potential dose delivered during blood feeding, 101–105 pfu.31,37 Therefore it is possible that the variability observed in our experiments results at least in part from variability in the dose delivered by donor mosquitoes.

Ultrastructural analysis of Cx. p. quinquefasciatus salivary glands infected with WNV has identified large organized clusters of virions or paracrystalline arrays and the association of virus with an as-yet unidentified salivary component (USC) (Figure 3A).30 Similar crystalline arrangements of St. Louis encephalitis virus in Cx. p. pipiens infected salivary glands have been observed and in some instances estimated to contain upwards of 50,000 virions.41 Virus aggregates have also been observed by TEM in saliva harvested from infected mosquitoes (Figure 3B). It is possible that the salivation of viral aggregates influences the temporal availability, the dose delivered by infected donor mosquitoes, and/or the dose of virus ingested by co-feeding recipient mosquitoes.

West Nile virus surveillance programs in the United States have identified at least 60 species of mosquitoes capable of being infected, an unprecedented number for any arbovirus. The potential for specific mosquito species to serve as WNV bridge vectors has been assessed based on factors such as feeding behavior, laboratory competence, and field isolations of virus.8 We evaluated the potential for NVT of WNV to facilitate infection of a potential bridge vector species. NVT of WNV from donor Cx. p. quinquefasciatus to a recipient Ae. albopictus mosquito was observed and resulted in the development of a high titer infection (7.52 log10 TCID50/mL), indicative of a disseminated infection. This indicates that NVT of WNV can facilitate infection of mosquitoes that may potentially transmit to human or equine hosts.

Assessment of the contribution of avian species to the maintenance/amplification of WNV has been based on level and duration of viremia.42,43 These analyses ignore the possibility that after the bite of an infected mosquito some vertebrates may experience a transient concentration of circulating virus in the blood. If NVT of WNV is dependent on the dose and temporal availability of virus that the donor inoculates, then it seems logical to assume that small vertebrates such as rodents and potentially small bird species would have a higher probability of supporting NVT in nature because the higher blood volumes in larger animals would serve to dilute the concentration of circulating virus. Observations of variability in host bloodmeal source of Culex spp. mosquitoes,44,45 seasonal changes in feeding behavior,46 coupled with potential attack rates,15 and high WNV field infection rates47 appear conducive for NVT to occur in nature. Further studies of the availability of virus immediately after the bite of an infected mosquito are necessary to assess the relative contribution of “dead-end” hosts to WNV maintenance and transmission.

Table 1

Nonviremic transmission of West Nile virus between Culex pipiens quinquefasciatus donor mosquitoes and recipient mosquitoes at increasing increments of spatial separation

Co-Feed# FeedsDonors engorged per feedAverage donor titer*Serum titer*†Total recipientsRecipients infected/nAverage recipient titer*Average % infected‡
* Virus titers calculated as log10 TCID50/mL.
† Serum titer only reported if observed in conjunction with positive recipient mosquitoes.
‡ Calculated as the average percent infection for only those feeds in which positive recipients were observed.
¶ Indicates recipient mosquitoes were allowed to feed at the same exposure site as donor mosquitoes immediately following 5 min of donor feeding.
Spatially restricted¶5≤27.5 ± .57Undetected < 13111/64
 1/72
 1/626.14 ± .331.52%
    10 mm6≤ 27.56 ± .31Undetected < 12320N/A0.00%
    20 mm5≤ 27.64 ± .452.952296/495.18 ± .2212.24%
    30 mm6≤ 37.78 ± .41Undetected < 12900N/A0.00%
    40 mm517.63 ± .22Undetected < 13141/486.952.08%
    50 mm5≤ 27.79 ± .45Undetected < 12900N/A0.00%
Ae. albopictus517.61 ± .19Undetected < 13191/647.521.56%
Table 2

Nonviremic transmission of West Nile virus between temporally separated Culex pipiens quinquefaciatus mosquitoes

Co-FeedDonor titerDonors engorgedSerum titer*Recipients infected/n (%)Average recipient titer*
* Virus titers calculated as log10 TCID50/mL.
15 min6.521Undetected < 12/68 (2.94)5.2 ± 1.41
5.951Undetected < 10/58 (0.00)N/A
30 min6.521Undetected < 10/70 (0.00)N/A
45 min7.521Undetected < 11/84 (1.19)4.52
60 min6.521Undetected < 10/66 (0.00)N/A
Figure 1.
Figure 1.

Spatial separation of donor and recipient mosquitoes feeding on a mouse.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 3; 10.4269/ajtmh.2007.76.424

Figure 2.
Figure 2.

Quantitative reverse transcription-polymerase chain reaction of West Nile Virus (WNV)-infected salivary glands dissected from intra-thoracically infected Culex pipiens quinquefasciatus mosquitoes over a 21-day time course of infection (•). PFU = plaque-forming units. Error bars represent standard deviation.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 3; 10.4269/ajtmh.2007.76.424

Figure 3.
Figure 3.

A, West Nile virus within a Culex, p. quinquefasciatus saliva cavity, 28 days post-infection (d.p.i.). Virus particles (Vp) line the saliva (Sa) cavity along the epithelial cell membrane and surround an unknown salivary component (USC). Cy = cytoplasm; Mi = mitochondrion; Va = vacuole. B, negative stain of mosquito saliva 15 d.p.i. Inset, high-power magnification of an individual virus aggregate. Arrows = single virus particles; arrowheads = virus aggregates; pSa = grainy, dark-staining areas putatively identified as mosquito saliva.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 3; 10.4269/ajtmh.2007.76.424

*

Address correspondence to Stephen Higgs, Department of Pathology, Keiller 2, 104, 301 University Blvd., Galveston, TX 77539-0609. E-mail: sthiggs@utmb.edu

Authors’ addresses: Charles E. McGee, Bradley S. Schneider, Yvette A. Girard, Dana L. Vanlandingham, and Stephen Higgs, Department of Pathology, University of Texas Medical Branch, Keiller 2, 104 301 University Blvd., Galveston, TX 77539-0609.

Acknowledgments: C. E. McGee, B. S. Schneider, and Y. A. Girard were supported by the Centers for Disease Control Fellowship Training Program in Vector-Borne Infectious Diseases T01/CCT622892. D. L. Vanlandingham was supported by National Institutes of Health T32 Grant (A107536). The authors are thankful for the technical assistance of Jing H. Huang for the rearing of the Cx. p. quinquefasciatus and Ae. albopictus mosquitoes and the generous contribution of mice by Dr. Richard B. Pyles. This work was supported by funds provided by the University of Texas Medical Branch Department of Pathology.

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