Dengue virus type 2: replication and tropisms in orally infected Aedes aegypti mosquitoes
© Salazar et al. 2007
Received: 11 September 2006
Accepted: 30 January 2007
Published: 30 January 2007
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© Salazar et al. 2007
Received: 11 September 2006
Accepted: 30 January 2007
Published: 30 January 2007
To be transmitted by its mosquito vector, dengue virus (DENV) must infect midgut epithelial cells, replicate and disseminate into the hemocoel, and finally infect the salivary glands, which is essential for transmission. The extrinsic incubation period (EIP) is very relevant epidemiologically and is the time required from the ingestion of virus until it can be transmitted to the next vertebrate host. The EIP is conditioned by the kinetics and tropisms of virus replication in its vector. Here we document the virogenesis of DENV-2 in newly-colonized Aedes aegypti mosquitoes from Chetumal, Mexico in order to understand better the effect of vector-virus interactions on dengue transmission.
After ingestion of DENV-2, midgut infections in Chetumal mosquitoes were characterized by a peak in virus titers between 7 and 10 days post-infection (dpi). The amount of viral antigen and viral titers in the midgut then declined, but viral RNA levels remained stable. The presence of DENV-2 antigen in the trachea was positively correlated with virus dissemination from the midgut. DENV-2 antigen was found in salivary gland tissue in more than a third of mosquitoes at 4 dpi. Unlike in the midgut, the amount of viral antigen (as well as the percent of infected salivary glands) increased with time. DENV-2 antigen also accumulated and increased in neural tissue throughout the EIP. DENV-2 antigen was detected in multiple tissues of the vector, but unlike some other arboviruses, was not detected in muscle.
Our results suggest that the EIP of DENV-2 in its vector may be shorter that the previously reported and that the tracheal system may facilitate DENV-2 dissemination from the midgut. Mosquito organs (e.g. midgut, neural tissue, and salivary glands) differed in their response to DENV-2 infection.
Dengue is an arthropod-borne viral disease of major public health importance worldwide. Currently, the dengue pandemic affects more than 100 countries, including nations in Africa, the Americas, the Indian subcontinent, Southeast Asia, the Eastern Mediterranean, and the Western Pacific . The World Health Organization (WHO) estimates there are 50 million annual cases of dengue infection worldwide, about 500,000 hospital admissions, and 22,000 dengue-related deaths. Globally, more than 2.5 billion people are at risk for this disease. The emergence of dengue in the Americas has been especially dramatic [2, 3].
Dengue virus (DENV) is comprised of four closely related, but antigenically distinct serotypes (DENV-1, DENV-2, DENV-3 and DENV-4), multiple genotypes enclosed in each serotype . The Aedes aegypti mosquito is the primary vector for all four serotypes. The mosquitoes become infected after feeding on a viremic individual, then a reported EIP of 7–14 days is required before the mosquitoes can transmit the virus to a new host [2, 5]. Environmental factors (including temperature and humidity) as well as intrinsic factors (such as vector competence and viral genotype) affect the EIP .
Host organ and cell susceptibility and permissiveness condition virus tropisms in an organism . A number of studies have investigated DENV-vector interactions [8–14], and the important role of virus genotype in efficient Aedes aegypti infection has been revealed . In this study, we wanted to determine the virogenesis of DENV-2 infection in recently colonized vector mosquitoes from a dengue endemic area, which previously had been shown to be very susceptible to DENV infection .
In the natural course of infection for the virus, DENV is ingested in a blood meal, then it encounters and must overcome midgut infection and escape barriers , which vary among mosquito populations. Infection of mosquito midguts is known to occur in a dose-dependent manner . Elucidation of the kinetics of replication and tropism of virus in recently colonized (field relevant) Aedes aegypti mosquitoes would provide a better understanding of transmission potential and vector-virus interactions that condition dengue epidemiology and epidemic potential. In this study, we determined: 1) the kinetics of DENV infection in orally infected mosquitoes, 2) the DENV-2 tropisms for different mosquito tissues and organs, 3) a possible anatomic explanation for virus dissemination from the midgut, and 4) the minimal time required for infection of salivary glands in recently colonized Aedes aegypti mosquitoes from Chetumal, Mexico and in a long colonized or a selected Aedes aegypti strain.
The presence of DENV-2 antigen in the tracheal system correlates with early dissemination in Chetumal mosquitoes
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Each of the mosquito strains examined were susceptible to DENV-2. However, the Chetumal and D2S3 strains were clearly more susceptible to DENV-2 infection than the Rex-D mosquitoes (Figure 1). DENV-2 replication in infected Rex-D mosquitoes was also slower than in Chetumal mosquitoes (data not shown). Unfortunately, we did not have a long colonized strain of Chetumal mosquitoes to compare to the newly colonized strain. Colonization has been shown to select for certain genotypes in Aedes aegypti . Thus the use of long-colonized mosquitoes as well as high passaged viruses from an area could confound the interpretation of the actual epidemic potential of DENV in that area. It would be prudent to study vector-pathogen interactions using field-relevant mosquitoes as well as low passage viruses from a specific area in order to draw more precise conclusions about the risk of dengue transmission in that area.
It was surprising that the Chetumal mosquitoes were as permissive to DENV-2 productive infection as the D2S3 strain, which was selected for DENV susceptibility . The susceptible phenotype is likely conditioned by different genetic mechanisms, which remain to be determined. However, the results do confirm previous studies demonstrating that mosquitoes from the eastern portion of the Yucatan Peninsula (an endemic area for dengue) are remarkably competent vectors for DENV .
DENV-2 infection was detected in midgut epithelial cells as early as 2 dpi. Viral titers and virus antigen then increased until 10 dpi when both virus titer and antigen decreased (Figure 2 and 3). In contrast, Q-PCR revealed that viral RNA copy number remained fairly constant even late into the EIP (Figure 3). Conversely, DENV-2 antigen was not cleared from salivary glands or neural tissues at equivalent time points. The former observation is of interest, because once a mosquito is productively infected with an arbovirus, she is capable of transmitting the virus for life. Obviously, any virus modulation in the salivary glands would be epidemiologically significant since transmission efficiency could be reduced. Modulation of virus in the midgut is provocative; either the virus or the epithelium is active in modulating the virus infection. The continued presence of DENV-2 RNA suggests a probable mechanism of persistence in vectors. DENV-2 as other arboviruses can persistently infect insect and human cell lines and arthropod vectors [20–23]. It is unlikely that the modulation is conditioned by the metabolic activity of the midgut epithelium, because a second uninfectious bloodmeal provided 14 days after DENV-2 infection of Aedes aegypti failed to reestablish virus production in the midgut (data not shown). The decline in infectious virus and viral antigen may be associated with an antiviral response, post-transcriptional or post-translational repression, or other self-limiting factors such as epithelial cells physiological status (i.e. cell aging). Further studies will be necessary to elucidate the molecular determinants of this phenomenon.
The mechanism(s) by which DENV disseminates from the midgut are not well understood. This study suggests that the mosquito tracheal system may be implicated in DENV dissemination (Figure 4 and Table 1). The presence of virus antigen in tracheal system significantly correlated with DENV-2 dissemination, mostly between 3 and 5 dpi. Tracheoles are part of the tracheal system, are 0.2 to 1 μm in diameter, are fluid-filled, and reach every tissue in the insect. The tracheal system has been reported to act as dissemination conduit for different viruses in insects. Notable examples are Autographa californica M nuclear polyhedrosis virus (AcMNPV) infecting Trichoplusia ni, nucleopolyhedrovirus infecting Bombyx mori, and Sindbis virus in Aedes albopictus [24, 25]. The tracheal system is also suspected to be the midgut escape conduit for Venezuelan equine encephalitis virus (VEEV) in Ochlerotatus taeniorhynchus . However, this is the first report indicating that DENV may use the mosquito tracheal system during dissemination.
Unlike a number of other arboviruses, DENV displayed no tropism for midgut-associated (Figure 6-C) or flight muscles. Rift Valley Fever virus infects both skeletal and visceral muscles . Sindbis virus replicates in visceral muscles of Aedes albopictus . West Nile virus infects the contiguous muscles of the posterior and anterior midgut in Culex mosquitoes . The reasons for mosquito muscle refractoriness to DENV are unknown and require further analysis. Rift Valley fever virus and Sindbis alphavirus exhibit tropisms for cardia tissue [11, 30], while DENV-2 was not routinely found in this tissue.
Interestingly, DENV-2 infection was detected in 36% of Chetumal mosquito salivary glands by 4 dpi. In contrast, < 15% of Rex-D mosquitoes had infected salivary glands at 7 dpi and salivary gland infection rates >36% weren't detected until 10 dpi. The Chetumal mosquitoes would seem to be more vector competent than the Rex-D mosquitoes. Reportedly the EIP for DENV is between 7 and 14 days, and it can be affected by conditions such as temperature, humidity and viral dose [2, 5, 6, 31]. Blood meals provided to mosquitoes in our studies contained 1.7 ± 0.7 × 107 PFU/ml, which is likely biologically relevant, since viremia titers of DENV-2 in humans may vary from 102 to 107 PFU/ml or 103 to 108 TCID50/ml [32, 33]. In our observations, the lateral distal lobes were frequently the first site to become infected in the salivary glands, the medial and proximal lobes developed infection later (Figure 5). This pattern probably results from the higher virus receptor concentration in this region of the salivary glands, but this remain to be determined. The lateral distal lobes participate prominently in the secretion of enzymes and proteins involved in hematophagy [34, 35], therefore infection of this region might promote early virus transmission. Other studies have demonstrated that DENV-2 can be transmitted by mosquitoes with < 25% salivary gland tissue infected, if infection occurred in lateral lobes . Thus, our results suggest that the EIP for DENVs may be shorter than the commonly reported (at least with some strains of the vector). There is no sensitive and reliable laboratory animal model for dengue, and currently used in vitro assays for transmission may fail to detect small but transmissible amounts of virus at earlier times. However further studies will be necessary to demonstrate that infectious virus is being transmitted early in the EIP and that the results can be extrapolated to low passaged DENV strains.
A shorter EIP for dengue would have important epidemiological consequences in dengue transmission. In nature, Aedes aegypti female mosquitoes feed frequently and almost exclusively on humans, which confers greater fitness than feeding on other hosts . These highly anthropophilic mosquitoes may ingest 2 to 3 bloodmeals during a single gonadotropic cycle, feeding with a frequency of 0.63 to 0.76 times a day . Aedes aegypti mosquitoes, with a very short EIP, persistently infected salivary glands, and the ability to feed multiple times within a gonadotropic cycle, would seem to be the ultimate DENV vector, which could help explain the epidemic potential of dengue.
The results with the recently colonized Chetumal mosquitoes indicate that the EIP for DENVs may be shorter than the commonly reported, which could have important epidemiological consequences in dengue transmission. The mosquito tracheal system appears implicated in virus dissemination from the midgut, and viral infection in the vector is differentially modulated in different organs. DENV-2 exhibits differences in certain tissue tropisms in its vector in comparison to other arboviruses in their respective vectors.
The newly-colonized Aedes aegypti Chetumal strain was characterized for its vector competence for DENV-2 [16, 38]. Chetumal mosquitoes (F3 - F6) were originally collected from in the Yucatan Peninsula, Mexico. Mosquitoes were hatched and reared as reported previously [16, 17, 38]. After hatching female pupae were separated from males by size, since males are significantly smaller; transferred in plastic cups to cardboard cartons and allowed to emerge. Adult mosquitoes were maintained at 28°C, 70–80% relative humidity, with a 12–12 h light-dark photoperiod. Mosquitoes used as controls in tropism experiments were the Puerto Rico-Rex-D strain (a long colonized laboratory strain)  and the D2S3 strain (a laboratory selected, highly susceptible mosquito strain lacking a midgut escape barrier) .
A high passage DENV-2 strain (Jamaica 1409)  was obtained from the Centers for Diseases Control and Prevention (CDC- Fort Collins, CO, USA). The virus had been routinely passaged (> 25 times) in C6/36 cell culture, but there were only two relevant changes in comparison to the low passaged virus as the full-length sequence analysis indicated . These changes were a conservative amino acid substitution in E6 protein (Ile→Met) and a nucleotide change in the 3'UTR143 (G→T). The C6/36 cell cultures were infected at a multiplicity of infection (MOI) of 0.001 and incubated at 28°C for a week. At day 7, the medium was replaced with fresh medium, and at 12 days post-infection virus was harvested to prepare infectious bloodmeals [16, 42].
Aedes aegypti mosquitoes were challenged with DENV-2 Jam1409 using an artificial bloodmeal . Briefly, 100–150 Aedes aegypti female mosquitoes, 4 - 7 days post-emergence, were deprived of sugar for 24 h and water for 8 h prior to the bloodfeeding. The virus-infected cell monolayers were scraped from the flask surface, and 2 ml of this suspension was added to an equal amount of washed sheep erythrocytes to prepare the infectious bloodmeal, which was provided to the mosquitoes using a membrane feeding device. The meal was maintained at 37°C by circulating warm water though an external jacket. Mosquitoes were not allowed to feed longer than 45 min to prevent a decline in viral titers. Blood meals contained viral titers of 1.7 ± 0.7 × 107 PFU/ml. Blood fed mosquitoes were separated using a chill table, transferred to clean cartons, and maintained at as noted above.
To determine viral tropisms, mosquito (n = 30–40) tissues were examined for the presence of virus antigen after dissecting midguts and salivary glands and squashing their thoraxes, heads, and abdomens on slides.
Midguts were dissected in PBS and fixed in 4% paraformaldehyde (Electron Microscopy, Hatfield, PA) for at least 4 h. The fixative was then removed, rinsed once in PBS, and then incubated for 1 h at room temperature with constant rocking in a solution containing: 1× PBS-Ashburner's solution (13 mM NaCl, 0.7 mM Na2HPO4, 1 mM NaH2PO4 at pH = 7.2), 1% Bovine Serum Albumin, and 0.1% Triton X-100, called PBT-0.1 (PBS-BSA-Triton). Typically to detect DENV-2 antigen by IFA, the primary antibody was the serotype-specific mouse 3H5–1 monoclonal antibody (ATCC number HB-46). To confirm the decline in DENV-2 antigen in midguts, a polyclonal human anti-dengue specific antibody (Biodesign International, Saco, ME) was also used as the primary antibody. Midguts were then incubated with a 1:400 dilution of goat-antimouse antibody conjugated with Alexa-488 fluorochrome (Molecular Probes Inc, Eugene, OR). Midguts were washed twice with PBT-0.1 with constant rocking at room temperature for 2 hr and incubated with a 1:40 dilution of Phalloidin-Alexa 546 (Molecular Probes Inc, Eugene, OR) for 20 min. Midguts were rinsed with PBS and placed onto slides, covered with Vectashield® (Vector Laboratories Inc, Burlingame, CA), and a coverslip was sealed with nail polish on the slide. Preparations were examined using the Olympus BH2-RFCA epifluorescence microscope or a confocal microscope (FVX-IHRT Fluoview Confocal, LSM, Olympus).
Salivary glands were dissected in PBS onto Vectabond® (Vector Laboratories Inc, Burlingame, CA) pretreated slides, fixed in cold acetone or fixed in 4% paraformaldehyde- 2% glutaraldehyde, and maintained at 4°C until immunosassay. Abdomens, thoraxes and heads were squashed on glass Vectabond®-pretreated slides, fixed in cold acetone for 15 min, and kept at 4°C
IFA was used to detect DENV-2 antigen in salivary glands, thoraxes, abdomens and heads. The primary serotype-specific monoclonal DENV-2 antibody 3H5-1 was diluted 1:200 in PBS and incubated for 1 h on the tissue. Slides were then washed twice for 5 min with PBS, and goat-anti-mouse avidin-conjugated secondary antibody containing 0.01% Evans Blue (1:200; Amersham Pharmacia Biotech, Piscataway, NJ) was added. After 40 min, samples were washed 2× in PBS and incubated for 20 min with a 1:200 dilution of fluorescein-streptavidin (Amersham Pharmacia Biotech, Piscataway, NJ). Following two additional PBS washes of 5 min each and one with distilled water, DABCO oil was added to the tissues and coverslips were added. All the incubations were performed at 37°C. An Olympus BH2-RFCA fluorescence microscope was used to examine slides.
The midgut infection rate (MIR) was determined as the number of midguts containing DENV-2 antigen divided by the number of midguts examined. The dissemination rate (DR) was determined as the number of mosquitoes with detectable DENV-2 antigen in non-midgut tissues (eg, head squash tissues, salivary glands, fat body, etc.), divided by the number of mosquitoes with detectable virus antigen in the midgut.
Viral titers in midguts were determined by end point titrations (TCID50) in C6/36 cells. Titers were established using the microtiter plate titration assay described by Schoepp and Beaty , and endpoints were calculated by the Karber method  and expressed as log10 TCID50/ml. To determine viral titers in whole mosquitoes as well as in some midguts, plaque assay titrations in LLC-MK2 cells were used modifying the protocol originally reported by Malewicz and Jenkin .
DENV-2 genome copy number was determined Q-PCR in homogenized and filtered tissue from individual mosquitoes or midguts . RNA for PCR assays was extracted using QIAamp® Viral RNA Kit (Qiagen Sciences, Maryland, MA). The reaction mixture was prepared with 1× DyNAzyme® buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mix, 0.5× SYBRGreen I (Molecular Probes Inc, Eugene, OR), 2.5 mM MgCl2, 10 μM of forward and reverse primers (5'CAAGTCGAACAACCTGGTCCAT3' and 5'GCCGCACCATTGGTCTTCTC3' respectively), 0.4U DyNAzyme II Recombinant DNA Polymerase (Finzymmes, Espoo, Finland), and 2 μl cDNA in a final volume of 20 μL. The SYBR Green I 10,000× stock was diluted in DMSO to a working solution of 100×, which was stored at -20°C in 15 μL aliquots to minimize exposure to light and freeze-thaw cycles. Reactions were performed in a microseal 96 microplates covered with optically clear caps (MJ Research, Waltham, MA) and placed in a Opticon 2 thermal cycler. Settings were: 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, 64°C for 20 sec, 72°C for 30 sec, and 84°C for 1 sec for fluorescence measurement. After a final extension at 72°C for 10 min, a melting curve was ascertained using the program: 70°C to 95°C, 0.2°C/read, 1 sec hold to confirm product specificity.
The primers used targeted a 177 bp region of the DENV-2 NS5 gene and quantified (+) strand DENV-2 RNA in the sample. Standard curves were generated on each plate by analyzing 2 × 102 to 2 × 108 copies/reaction of DENV-2 from plasmid standards.
This research was supported by NIH AI45430 and AI48740. MIS was granted a Fogarty International Center scholarship (TW1130).
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