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    Reproducibility of DENV-2 quantitative real-time RT-PCR assays. The CV of three CT estimates was calculated among plasmid standards analyzed on the same plate. These CVs were then compared with the CVs of CT among the same plasmid standards but analyzed on different plates on different days.

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    Total RNA estimated with the RiboGreen assay. RNA from 10 midguts was quantified on each day. Whisker caps represent 10th and 90th percentiles. Box limits represent 25th and 75th percentiles. Lines within the boxes mark the median. Circles indicate the 5th and 95th percentiles. Day 0 corresponds to mosquitoes dissected immediately after they blood-fed.

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    Plaque assay vs. DENV-2 RNA copies with five different DENV-2 genotypes grown in C6/36 cells. The regression equation is DENV-2 copies = 0.242 PFU + 9.09 (R2 = 0.91). Both slope and y-intercept were significantly > 0 (P ≤ 0.0001). Line shown in the lower right-hand side of the graph is the RNA copy = PFU line.

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    Plaque assay vs. DENV-2 RNA copies in two replicate groups of D2S3 Ae. aegypti mosquitoes. The regression equations are DENV-2 copies = 0.358 PFU + 6.01 (R2 = 0.45) and DENV-2 copies = 0.335 PFU + 5.73 (R2 = 0.36).

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    Quantitative analysis of DENV-2 replication in the midgut of individual Aedes aegypti (Chetumal strain). Y error bars represent the standard deviation of DENV-2 RNA copies in the mosquitoes testing positive on each day. Bars indicate percentage of mosquitoes with a disseminated infection. Refer to Table 3 for number of positive midguts at each time point. Day 0 corresponds to mosquitoes dissected immediately after they blood-fed.

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    Quantitative analysis of DENV-2 replication in leg tissue from individual Aedes aegypti (Chetumal strain). Y error bars represent the standard deviation of DENV-2 RNA copies in the mosquitoes testing positive for each day. Bars indicate percentage of mosquitoes with DENV-2 (+) RNA in legs. Table 3 lists the number of positive mosquitoes at each time point.

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    Comparison of DENV-2 (+) RNA in midguts and legs over the course of the extrinsic incubation period.

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QUANTITATIVE ANALYSIS OF DENGUE-2 VIRUS RNA DURING THE EXTRINSIC INCUBATION PERIOD IN INDIVIDUAL AEDES AEGYPTI

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  • 1 Department of Microbiology, Immunology and Pathology, Arthropod-borne and Infectious Diseases Laboratory, Colorado State University, Fort Collins, Colorado

Dengue virus-2 (DENV-2) RNA was quantified from the midgut and legs of individual Aedes aegypti at each of 14 days postinfectious blood meal (dpi) in a DENV-2 susceptible strain from Chetumal, Mexico. A SYBR Green I based strand-specific, quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed. The lower detection and quantitation limits were 20 and 200 copies per reaction, respectively. Amounts of positive and negative strand viral RNA strands were correlated. Numbers of plaque-forming units (PFU) were correlated with DENV-2 RNA copy number in both C6/36 cell cultures and mosquitoes. PFU were consistently lower than RNA copy number by 2–3 log10. Midgut levels of DENV-2 RNA peaked 8 dpi and fluctuated erratically between 6 and 9 dpi. Copies of DENV-2 RNA varied significantly among infected mosquitoes at each time point. Quantitative real-time RT-PCR is a convenient and reliable method that provides new insights into virus-vector interactions.

INTRODUCTION

Dengue viruses (genus Flavivirus, family Flaviviridae) include four antigenically distinct serotypes (DENV 1 to 4) transmitted among humans by Aedes mosquitoes, primarily Aedes aegypti. The changing epidemiology of DENV has spurred a global resurgence of both dengue fever (DF) and dengue hemorrhagic fever (DHF) with DF incidence estimated at 50 million to 100 million cases per year and DHF ranking as a leading cause of pediatric hospitalization and death.1

Vector competence refers to the intrinsic ability of an arthropod to become orally infected and to support virus replication, dissemination, and transmission.2 Understanding the relative vector competence of mosquitoes at the species, population, and individual levels is critical to the study of vector biology and the success of future vector-borne disease control programs. Aedes aegypti populations vary in their competence for DENV.313 This is due, in part, to the presence or absence of midgut infection and/or midgut escape barriers (MIB, MEB).9,12,14 Vector competence studies of DENV are typically qualitative in nature, involving the detection of viral antigens in the head or legs. The vector competence of the experimental group is then estimated as the proportion of tested mosquitoes that develop a disseminated infection.

Dengue viruses are single-stranded, positive sense RNA viruses. Replication involves three distinct RNA species: genomic (+) RNA, the antisense (−) copy of the genome, and partially double-stranded RNA replicative intermediates.1517 The latter two species are only present while the virus is replicating.18 Asymmetric strand replication results in a 1–2 log10 excess of (+) RNA compared with (−) RNA.19,20 Quantification of (+) and (−) RNA during the course of infection is useful in assessing the kinetics of viral infection and replication. This can be achieved using a two-step reverse transcription-polymerase chain reaction (RT-PCR) in which only the forward primer is added to synthesize cDNA from the (−) RNA strands. Conversely, only the reverse primer is used during the RT step for samples to be measured for (+) RNA. Both primers are then used in the subsequent PCR reactions to amplify the cDNA pools generated. This approach has been used to quantify DENV (−) RNA in human peripheral blood mononuclear cells in which the amount of DENV (−) RNA correlates with plasma viral load.20

Plaque assays and end-point titrations are the most accurate methods for quantifying infectious DENV.2125 However, these assays are laborious and time consuming and require at least a week for completion.26,27 Plaque formation from DENV infection can also be restricted by the phenotype and passage history of the virus strain and by the type and lot of cells used in the assay.11,23 Rapid advances in molecular biology have facilitated new approaches to evaluate quantitative aspects of vector competence. In particular, quantitative real-time RT-PCR based assays provide the sensitivity, speed, and statistical power to conduct high-throughput experiments. The precise quantitation of viral RNA from a sample of only 5 μL allows for the study of viral infection, replication, and dissemination in specific tissues in individual mosquitoes on a scale that was previously impractical. Of the two widely used real-time RT-PCR formats, SYBR Green I based assays are less expensive, more flexible, and less susceptible to false negatives due to single nucleotide polymorphisms in the probe sequence than assays that use TaqMan.28 Although a single point mutation in the probe region can reduce target detection by 47% in TaqMan assays, SYBR Green I is a nonspecific dsDNA binding dye that only requires design of oligonucleotide primers for PCR to measure fluorescence emission. When bound to dsDNA, the fluorescence of SYBR Green I is increased ~1,000-fold, providing a sensitivity equal to that of TaqMan.2830 Furthermore, the reaction is reversible, facilitating the post-run dissociation or melting curve analysis for confirmation of product identity. This step improves assay specificity.

Real-time RT-PCR assays have only recently been adapted to study virus-vector interactions.11,27,3137 Here we report the quantitation of DENV-2 RNA during the 14-day extrinsic incubation period in individual Ae. aegypti. A SYBR Green I based strand-specific, quantitative real-time RT-PCR assay was developed to quantify DENV-2 RNA from individual midgut and leg samples of Ae. aegypti. To test the validity of our assay, parallel plaque and real-time RT-PCR assays were conducted on infected C6/36 cell cultures and three groups of infected Ae. aegypti.

MATERIALS AND METHODS

Aedes aegypti rearing and virus preparation.

Eggs from the F5 generation of an Ae. aegypti collection from Chetumal, Mexico, were hatched and reared in a controlled environment at 27°C, 80% relative humidity, and 12-hour photoperiod. An initial assessment showed that 85% of mosquitoes exposed per os to DENV-2 (JAM1409) develop a disseminated infection. The D2S3 strain, previously selected for high susceptibility to DENV-2 (JAM1409), was used for the assay comparison experiments. Typically 95–100% of D2S3 mosquitoes develop a disseminated infection.13 Adults were kept in 1.9 L waxed cardboard containers with water and sucrose. A high-passage Jamaica 1409 strain (JAM1409) of DENV-238 was grown in the Aedes albopictus C6/36 cell line.9 In assessing the correlation between PFU and DENV-2 RNA amounts, four additional DENV-2 genotypes were compared: American/Asian (Yuc14757), Cosmopolitan (96Mer/BC17), Asian 2 (97 Acap/C932), and American (94QRoo/BC-139). Strains, sources, and passage history of these viruses are listed in Table 1.

Mosquito infections.

Sucrose and water were removed 36 hours prior to oral challenge. Water-jacketed membrane feeders were covered with hog intestine and placed on containers with 3- to 4-day-old mosquitoes. Defibrinated sheep blood was mixed with the C6/36 cell-DENV-2 suspension (not centrifuged) (1:1), and ATP was added at a final concentration of 1 mM. Mosquitoes were allowed to feed for 45–60 minutes on a 37°C blood meal with final infectious virus titer of 109.5 tissue culture infectious dose 50% (TCID50)/mL.40 Mosquitoes that failed to feed to repletion were discarded.

Mosquito dissection and RNA extraction.

Ten Ae. aegypti were collected daily, chilled, and dissected live in 20 μL of Tris buffered saline (10 mM Tris-HCl [pH 7.5], 150 mM NaCl) on a chill table at ~4°C. Midguts and legs were placed in separate 1.5-mL microcentrifuge tubes containing 100 μL of a denaturing guanidine isothiocyanate buffer (RLT lysis buffer, Qiagen, Valencia, CA) to inactivate RNAses. Samples were snap frozen and stored at −80°C. RNA was extracted using RNeasy total RNA extraction kits (Qiagen) and eluted in 50 μL of RNAase free water.

Quantitation of total RNA.

The RiboGreen RNA-Specific Quantitation Assay (Molecular Probes, Eugene, OR) was used to quantify total RNA extracted from each mosquito to assess variation in amounts of starting tissue and in RNA extraction efficiency.41 We modified the published protocol to enable RNA quantitation using the Opticon Monitor fluorescence detection platform (MJ Research, Waltham, MA) that was also used in the real-time RT-PCR assay.42 The Opticon Monitor was calibrated for a 10X RiboGreen dye solution (60 μL per well) prepared with 1X TE and the 3200X DMSO stock solution provided by the manufacturer. Sample RNA was treated with the DNAase provided with the RiboGreen kit. A baseline was generated for each run by detecting background fluorescence in wells with 51 μL per well of 10X dye. The program was set to 60 seconds at 37°C, followed by 5 cycles for 20 seconds at 37°C, each followed by a fluorescence reading. The detection protocol was paused, 9μL of the DNAase-treated RNA samples and standards were added to 51 μL of 10X RiboGreen in triplicate, and the run was completed with ten 20-second cycles at 37°C each followed by a fluorescence reading. Data was exported into Microsoft Excel where baseline values were subtracted from sample readings. Fluorescence of the RNA standards was regressed on RNA amounts to derive a standard curve for quantification of RNA in mosquito tissues.

Preparation of DENV-2 standards.

The NS5 fragment of DENV-2 (JAM1409) was amplified and cloned into the pCR 2.1 plasmid using the TA cloning kit (Invitrogen, Carlsbad, CA). Inserts were sequenced to confirm presence of the target fragment. The HiSpeed Plasmid Midi Kit (Qiagen) was used for large-scale preparation and purification of the DENV-2 NS5 plasmid. After quantification by UV spectrophotometry, the plasmid was diluted to 108 plasmids/μL for use in a 10-fold dilution series. Aliquots were stored at −80°C with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO).

cDNA synthesis.

DENV-2 specific primers targeted a 177-bp region of the NS5 gene and have proven reliable in real-time RT-PCR studies of DENV-2.37,43 This gene codes for the RNA-dependent RNA polymerase, the most conserved of the DENV proteins.17 Primer sequences were DENV-2 NS5 forward (5′-ACAAGTCGAACAACCTGGTCCAT-3′) and DENV-2 NS5 reverse (5′-GCCGCACCATTGGTCT-TCTC-3′). The mfold Web server (http://www.bioinfo.rpi.edu/applications/mfold/old/dna/)44 was used to avoid potential secondary structures that might reduce primer binding. First-strand synthesis reactions contained 0.625 μM either forward, reverse, or paired primers and 0.20 mM dNTP mix, 5 μL of template RNA, and DEPC treated H2O to a final volume of 10.9 μL. To minimize inhibition of SYBR Green I, DTT was excluded.45 Reactions were heated to denature dsRNA complexes (86°C for 15 minutes) and brought to 0°C on an Eppendorf PCR-Cooler (Eppendorf, Hamburg, Germany). A total of 4 μL 5X First-Strand Buffer and 0.1 μL SuperScript II Reverse Transcriptase (RT) (Invitrogen) were added to bring the reaction volume to 20 μL. cDNA was synthesized at 42°C for 50 minutes. To quantify samples for DENV-2 (−) RNA, only the forward primer was added to synthesize cDNA from the (−) RNA strands. Conversely, only the reverse primer was used during the RT step for samples to be measured for (+) RNA. Both primers were then used in the subsequent PCR reactions to amplify the cDNA pools generated from a single primer. The RT was inactivated with a 15-minute incubation at 95°C. Controls without primers were included to confirm RT inactivation.

DENV-2 quantitative real-time RT-PCR.

The PCR mixture contained 1X DyNAzyme buffer (Finnzymes, Espoo, Finland), 0.2 mM dNTP mix, 0.5X SYBR Green I, 2.5 mM MgCl2, 0.25 μM forward and reverse primers, 0.4 U DyNAzyme II Recombinant DNA Polymerase (Finnzymes), and 2 μL cDNA in a final volume of 20 μL. SYBR Green I was obtained in a 10,000X stock concentration, diluted in DMSO to a working stock of 100X, and stored at −20°C in 15-μL aliquots to minimize exposure to light and freeze-thaw cycles.

Reactions were run in Microseal 96 Microplates covered with optically clear caps (MJ Research, Waltham, MA) and spun at 3,000 rpm for 5 minutes. Opticon 2 thermal cycling settings were 95°C for 10 minutes, followed by 40 cycles of 95°C for 10 seconds, 64°C for 20 seconds, 72°C for 30 seconds, and 84°C for 1 second for fluorescence measurement. After a final extension at 72°C for 10 minutes, a melting curve was obtained with the program: 70°C to 95°C, 0.2°C/read, 1 second hold, and the negative first derivative of the dissociation rate (-dF/dT) was used to confirm product specificity. This protocol was used to measure both (+) and (−) DENV-2 RNA. Standard curves were generated on each plate by analyzing 2 × 102 to 2 × 108 copies/reaction of DENV-2 plasmid standards. All cDNA samples and plasmid standards were assayed in triplicate.

Fluorescence generally followed a logistic curve increasing with each PCR cycle. The point at which fluorescence had increased ~50% over background is the fluorescence threshold. This was subsequently set at 0.040 (−1.40 log10) for all assays. The cycle threshold (CT) is the number of cycles along the abscissa at which fluorescence crosses this threshold. Amplification efficiency (E) was calculated from the slope of the linear regression of CT versus the log10 template copy number where:

E=10(1/slope)1

An E of 1 corresponds to 100% efficiency. E was derived using a 10-fold dilution series of sample cDNA and DNA standards. Assay repeatability was estimated using the coefficient of variation (CV = [σx/x̄] × 100) among triplicate samples within a 96-well plate and among the same standards run on 10 different plates. To assess variation in the efficiency of cDNA synthesis, we estimated variation in the reverse transcription and PCR reactions separately.46 Triplicate cDNA synthesis reactions on DENV-2 positive and negative control mosquito RNA samples were performed followed by quantitative real-time PCR reactions in triplicate for each cDNA pool.

Plaque assays.

Plaque and real-time RT-PCR assays were performed in parallel on DENV-2 infected Ae. aegypti (D2S3) and C6/36 cells to assess the relationship between DENV-2 RNA copy number and numbers of infectious virus particles. Mosquitoes were infected with the high-passage JAM1409 strain, and the C6/36 cells were infected with one of five DENV-2 strains: JAM 1409, Yuc14757, 96Mer, 97 Acap, 94QRoo (Table 1). Frozen mosquitoes were thawed, triturated individually in 750–1,000 μL of minimal essential media (MEM) containing 2% fetal bovine serum (FBS) (Gemini Bio-Products, Woodland, CA), spun at 14,000 rpm for 3 minutes, and the supernatant was filtered through an 0.22-μm syringe filter. RNA was extracted from an aliquot of each sample using the QIAamp viral RNA extraction kit (Qiagen), and a separate aliquot was used for the plaque assay. Confluent cultures of LLC-MK2 cells were grown in MEM with Earl’s salts and supplemented with 10% FBS, 2 mM l-glutamine (Invitrogen, GIBCO Products (Carlsbad, CA), 1X nonessential amino acids solution (Cellgro) (Mediatech, Herndon, VA), 100 U/mL of penicillin, and 100 μg/mL of streptomycin, and maintained in a CO2 incubator at 37°C. Ten-fold dilutions of each sample were incubated for 1 hour on LLC-MK2 cell monolayers in 6-well plates with constant rocking. The first overlay was then added (1% agar, 1X Earl’s balanced salts solution, 0.066% yeast extract, 0.33% lactoal-bumin hydrolysate, 2% FBS, 0.22% sodium bicarbonate, 50 μg/mL of gentamicin, and 2 μg/mL of fungizone). After solidification of the initial overlay, 6-well plates were inverted and incubated for 7 days at 37°C in a CO2 incubator. A second overlay was then added containing 0.125% neutral red, and plaques were counted after 48 hours of incubation.

RESULTS

DENV-2 quantitative real-time RT-PCR.

Melting curve analyses were completed to assess the specificity of amplified products. A single -dF/dT peak occurred at ~86°C with a width of ~1.6°C. Estimates of the variation in CT values attributed to the cDNA synthesis and PCR steps are summarized in Table 2. Repeatability in CT values was compared among cDNA syntheses starting with RNA from three infected mosquitoes. Among three real-time PCR reactions all prepared from a single cDNA synthesis of an infected mosquito, the CV averaged 2.78% (range 1.71–3.59%, SD 0.58). Among three independent cDNA syntheses from a single mosquito, the CV averaged 2.1% (range 1.96–2.35%, SD 0.21). The CVs of the two steps did not differ significantly (P = 0.087). The high CT values from negative and no RNA controls were due to nonspecific fluorescence (likely primer-dimers) commonly observed with SYBR Green based assays.

The quantity of DENV-2 (JAM1409) NS5 plasmids contained in a 10-fold serial dilution was compared with the relative amount of cDNA contained in a 10-fold serial dilution as estimated by real-time PCR. This was done to test for nonlinearity when assessing quantity of DENV-2 (JAM1409) NS5 with plasmid standards as compared with cDNA. The slopes of plasmid amounts (−3.47 ± 0.16) and cDNA amounts (−3.28 ± 0.10) were not significantly different. The mean E of 0.94 for the DENV-2 DNA plasmid standards (N = 30, 102 to 108) was similar to 1.02 for the cDNA samples (N = 3, undiluted to 10−4).

The reproducibility of quantitative real-time PCR assays was assessed by calculating the CV of three CT estimates among plasmid standards loaded and analyzed on the same plate. These CVs were then compared with the CVs of CT among the same plasmid standards but loaded and analyzed on different plates on different days. The CV estimates were plotted against log10 plasmid copies/20 μL reaction (Figure 1). The accuracy of copy number estimates was reduced in samples with < 100 copies of DENV-2 plasmid, and nonspecific peaks were revealed in the melting curves of the 101 standard. The intraplate CV was 5.4% with 100 plasmid copies but then fell to ~1% with 2 × 103 to 2 × 107 copies. Among plates, CVs were slightly higher than CVs within plates. These results are comparable to other published real-time RT-PCR flavivirus assays.33,4749

RNA quantitation assay.

Mosquitoes differed significantly in midgut RNA levels within each time point and between daily means (ANOVA, P < 0.0001) (Figure 2). Total RNA levels in midguts over the 14 days postinfectious blood meal (dpi) ranged fourfold from ~500 to 2,000 ng/midgut. Total RNA from midguts presented a cyclical pattern with peaks at 1, 8, and 14 dpi and troughs at 0, 2–3, and 11–12 dpi. This fluctuating pattern suggests major metabolic changes in the midgut. Transcription during the first days was likely associated with blood digestion. Mosquitoes were not given a second blood meal nor was gonotrophic status recorded in individual females. The relationship of physiologic processes to total RNA fluctuations is therefore unknown.

Real-time RT-PCR and plaque assays.

Plaque and real-time RT-PCR assays were performed in parallel on DENV-2 infected C6/36 cell-culture samples and Ae. aegypti to assess the relationship between DENV-2 RNA and numbers of infectious virus particles. Among the 5 DENV-2 genotypes, the average rate of DENV-2 RNA (both strands)/PFU was 1,592 with a range of 135 to 4,019 (Figure 3). The regression curve lies above the RNA copy = PFU line. In general, viral RNA levels overestimated PFUs by ~2–3 logs. The slope of the untransformed data (ΔRNA/ΔPFU) was 80.0, suggesting that among the 5 DENV-2 genotypes there were 80 viral RNA copies/PFU or that 80 in 81 copies (98.8%) are noninfectious.

Figure 4 shows a similar regression in DENV-2 (+) RNA collected from DENV-2 infected D2S3 Ae. aegypti and fed upon DENV-2 virus preparations made on two different dates. Again, the regression curves lie above the RNA copy = PFU line and the y-intercepts were > 0 (P ≤ 0.0001). The slopes and y-intercepts were not significantly different between DENV-2 virus preparations. In the first infection, the average rate of DENV-2 (+) RNA/PFU was 367 with a range 72–2,147, while in the second infection the average rate of DENV-2 (+) RNA/PFU was 12,227 with a range 2,294–69,063. The slope of the untransformed data (ΔRNA/ΔPFU) varied from 59 to 2,954 (+) RNA copies/PFU, suggesting that ~98.3–99.9% of the viral (+) RNA measured by real-time RT-PCR is noninfectious.

Dengue virus extrinsic growth kinetics and strand-specific detection.

The amount of DENV-2 RNA (+) and (−) strands over the course of the extrinsic incubation period are shown in midguts in Figure 5 and in legs in Figure 6. DENV-2 RNA levels in midguts over the 14 dpi ranged 3 orders of magnitude from ~103 to 106 copies/midgut. In comparison, total RNA varied only fourfold (Figure 2), thus adjusting for total RNA had an imperceptible effect, and in the remaining analyses, neither DENV-2 RNA levels nor PFU are standardized on total RNA.

DENV-2 RNA (+) and (−) in the midgut decreased sharply during the “eclipse” phase of infection between 0 and 2 dpi. The detection of (−) DENV-2 RNA during this period probably reflects replicative intermediates released from C6/36 cells in the infectious blood meal. A replication phase with steadily increasing viral RNA levels was observed from 2 to 6 dpi. At this point, DENV-2 RNA replication in the midgut fluctuated from 6 to 9 dpi. Viral RNA copy number then steadily increased from 10 to 14 dpi. Viral RNA copy number varied significantly among mosquitoes sampled at each time point (Table 3). There was a consistent relationship between (+) and (−) RNA over the 14 days (r = 0.87 to 0.99 between 3 and 14 dpi) (Table 3).

In the disseminated infection (leg) assay (Figure 6), 1 of 10 mosquitoes tested positive for both strands on Day 1. At 4 dpi, a single mosquito demonstrated (+) but not (−) RNA (Table 3). After 4 dpi, the number of positive samples and the level of (+) RNA increased gradually. In comparison with (+) RNA, detection of (−) RNA was delayed and the rate of increase in (−) RNA was lower. There was a significant positive correlation between (+) and (−) RNA detected in legs after 8 dpi (Table 3). Prior to Day 8, there were not enough infected leg samples to test for a correlation. In contrast to midgut samples, (−) RNA was detected in a few of the (+) RNA positive legs until 12 dpi.

The amount of DENV-2 (+) RNA in midguts and legs over the course of the extrinsic incubation period are plotted in parallel in Figure 7. The amount of DENV-2 (+) RNA in legs was correlated with the dpi (r = 0.82, P ≤ 0.001). In contrast, the amount of DENV-2 (+) RNA in midguts was not correlated with the dpi (r = 0.48, P ≥ 0.05) and was only weakly correlated with DENV-2 RNA (+) strands in legs (r = 0.55, P ≥ 0.05). Furthermore, DENV-2 (+) RNA in midguts and legs were independent if data from 0 to 3 dpi were removed (r = 0.29, P ≥ 0.05).

DISCUSSION

SYBR Green I is a nonspecific dsDNA binding dye that only requires design of conserved oligonucleotide primers to measure rates of increase in fluorescence during PCR. SYBR Green I based assays therefore facilitate development of protocols for novel arboviral genotypes, serotypes, or species, in which information on target sequences may be limited. When bound to dsDNA, the fluorescence of SYBR Green I is increased ~1000-fold, providing the sensitivity required for assays of low starting copy number.29,30 The assay reported herein has a lower detection limit of 20 RNA copies and a lower quantitation limit of ~200 RNA copies. Lowered specificity is of potential concern with SYBR Green because it is not sequence specific, and low-level background fluorescence can arise from primer-dimers and other nonspecific amplicons. A melting curve analysis and agarose gel electrophoresis help to assess amplicon identity.

Quantification of housekeeping genes is commonly used to standardize the number of copies detected among samples that vary in overall RNA amounts (e.g., legs and midguts). This is problematic because expression of housekeeping genes varies among tissues, developmental and physiologic states, and experimental treatment.5052 Furthermore, during blood feeding and vitellogenesis, dramatic changes in overall transcription occur among many housekeeping genes. We measured total RNA to avoid specific expression profiles among housekeeping genes. At any time point, viral RNA represented a small fraction (< 1%) of the total RNA, however, vector RNA varied only fourfold over the course of the extrinsic incubation period while viral RNA fluctuated by three orders of magnitude. Therefore, adjustments of viral RNA relative to total RNA imperceptibly changed viral growth curves.

Among mean values from mosquitoes processed on each day, total RNA (Figure 2) and DENV-2 (+) RNA in the midgut (Figure 5) were correlated between Days 2 and 10 postinfection (r = 0.80, P = 0.009). Furthermore, in individual midguts over all days (N = 139), total RNA was correlated with DENV-2 (+) RNA (r = 0.31, P = 0.0001) and DENV-2 (−) RNA (r = 0.26, P = 0.0022). This suggests the possibility that metabolic changes in the midgut (reflected in changes in total RNA) also affect DENV-2 virus replication. For example, both total and DENV-2 (+) RNA peak on Day 8 (Figures 2 and 5). This supports previous findings that suggest that the midgut has less nutrient availability after 8 dpi and this may lead to a decrease in virus replication.32 However, when individual midguts were analyzed by day, total and DENV-2 (+) RNA in individual mosquitoes were only correlated on Days 0, 12, and 13. This loss of correlation suggests that variation measured among individual mosquitoes on a given day was much larger than the variation among means on different days.

Positive correlations between DENV-2 (+) RNA and numbers of PFU were observed in both C6/36 cells and Ae. aegypti (Figures 3 and 4). The large ratio of RNA copies to PFU reflects the complex and dynamic relationship between a replicating virus and the cells it infects. The variability of infectivity estimates arises due either to defective, immature, or inactivated virus particles or to free viral RNA from within cells harboring an infection. Other factors could include numbers of freeze-thaw cycles or fluctuating pH among samples. The RNA copy:PFU ratios published here are comparable to those observed for yellow fever virus in Ae. aegypti (1,000–5,000 RNA copy/PFU),27 those reported for West Nile virus in saliva of various mosquito species (80–1,134,649 genome equivalents/PFU),53 and those reported for DENV-2 in peripheral blood mononuclear cells (10–200 RNA copies/PFU).20

Studies of vector competence have generally reported the proportion of exposed mosquitoes that develop midgut or disseminated infections or transmit the virus to suckling mice.13,5458 This is the first study to examine fluctuations in DENV-2 RNA copy number in the midgut and legs among individual mosquitoes over the 14-day course of the extrinsic incubation period. A typical eclipse phase associated with decrease in virus levels during digestion of the blood meal was seen on Days 0–2. During the eclipse phase, there was a poor correlation between amounts of (+) and (−) strands from 0 to 3 dpi. This may reflect a variable strand ratio in the blood meal. Analyses of DENV-2 (+) and (−) RNA in five independently prepared blood meals revealed significant variation in the (+) to (−) strand ratio (1.26–1.55) with no correlation between the amount of the two strands (r = 0.0014).

One mosquito developed a disseminated infection during the eclipse phase. This has been observed in earlier studies, and the prevailing hypotheses involve either a “leaky midgut” and/or tracheal cell conduits that enable rapid viral escape from the midgut.2,5964 This could also arise from variation in tracheal cell density, location, or degree of basal lamina penetration.62 Dissemination rates first reached 50% at 6 dpi. Detection of (+) RNA in legs preceded detection of (−) RNA, and the number of mosquitoes testing positive for (+) RNA exceeded those with (−) RNA until 10 dpi (Table 3). Our strand-specific assay thus detected early dissemination of virus prior to initiation of replication. The steady increase in viral RNA between 2 and 6 dpi suggests that there is no abrupt increase in virus when midgut epithelial cells become infected and replication begins. Antiviral or other mosquito cell responses might have caused the pronounced and rapid changes in viral RNA copy number observed in the midgut between 6 and 9 dpi. A similar decrease in DENV-2 levels in Aedes albopictus (whole bodies, 10 dpi) has been observed.65

This is the first documentation of pronounced fluctuations of DENV-2 in individual midguts. The standard errors around estimates of DENV-2 RNA amounts were large—reflecting the variation in DENV-2 RNA levels among mosquitoes. Nevertheless, between 6 and 10 dpi, day-by-day comparison of the amounts of DENV-2 RNA were significant (Duncan’s multiple range and Student-Newman-Keuls tests not shown). During 6–10 dpi, the percentage of mosquitoes with disseminated infections was also increasing but with many fluctuations (Figure 5). It is possible that the same mechanisms affecting DENV-2 RNA may also condition mid-gut escape or dissemination barriers. This would be especially true if viral dissemination from the midgut is dose dependent with a mass exodus of mature virus occurring once a threshold is reached. The rebound in DENV-2 RNA seen on Day 8 may be a viral response to a putative antiviral response (Figure 5). Accumulation of dsRNA or viral proteins that modulate RNAi or apoptosis at certain levels may facilitate this rebound.6668

Real-time RT-PCR appears to be a practical and reliable method that may provide new insights into virus-vector interactions during the extrinsic incubation period. We are in the process of repeating this study in lines of Ae. aegypti that have been selected for a low midgut infection rate (Ibo 11)58 and a low dissemination rate (D2MEB).9 The precise quantitation of viral RNA during infection, replication, and dissemination in specific tissues may help elucidate how DENV refractory Ae. aegypti reduce or eliminate DENV infections.

Table 1

Source and passage history of the DENV-2 strains used in this study

GenotypeIDSourceYearPassage history
* Phylogenetic analysis on group genotypes was based on the prM-E viral region (Lorono-Pino and others,39). The DENV-2 Yuc 14757 strain is a new isolate not included in the original phylogenetic analysis.
American94 QRooHuman isolate (BC-139) from Mexico. Acquired by the Centers for Disease Control and Prevention.1994Patient → C6/36 → 2 (C6/36)
American/AsianYuc 14757Human isolate (14757) from the Centro de Investigaciones Hideyo Noguchi, Merida, Yucatan, Mexico.2002Patient → C6/36 → BHK → C6/36
Asian 297 AcapHuman isolate (C932) from the Instituto Nacional de Salud Publica, Cuernavaca, Mor., Mexico.1997Patient → C6/36 → 3 (C6/36)
Cosmopolitan96 MerHuman isolate (BC-17) from Mexico. Acquired by the Centers for Disease Control and Prevention.1996Patient → C6/36 → 2 (C6/36)
PrototypicJam 1409Laboratory prototypic DENV-2 strain initially isolated from a patient in Jamaica.1983High passage
Table 2

Comparison of variation in cDNA synthesis and amplification steps

Mosquito no.*RNA aliquotCTqPCR CV‡ (%)CT§RT CV (%)
CT, cycle threshold; CV, coefficient of variation.
* Numbers represent individual infected mosquitoes. Letters represent aliquots of RNA used in the cDNA synthesis step.
† Mean cycle threshold values ± standard deviation of three wells with the same cDNA sample.
‡ Coefficient of variation.
§ Mean cycle threshold values ± standard deviation of three different cDNA samples from the same mosquito. Fluorescent signals from negative and no RNA controls were nonspecific (likely primer-dimers) according to melting curve analysis.
¶ ANOVA of CV values of the two steps was not significant (P = 0.087).
A20.32 ± 0.482.38
1B20.70 ± 0.623.0220.26 ± 0.482.35
C19.75 ± 0.472.39
A18.80 ± 0.683.59
2B19.28 ± 0.603.1219.21 ± 0.381.96
C19.54 ± 0.643.25
A20.27 ± 0.502.47
3B20.79 ± 0.361.7120.72 ± 0.422.03
C21.11 ± 0.653.09
Mean¶2.78 ± 0.582.11 ± 0.21
A34.24 ± 1.534.46
Negative controlB31.84 ± 0.310.9632.61 ± 1.414.31
C31.77 ± 0.040.11
A33.92 ± 1.243.66
No RNA controlB32.52 ± 0.060.2033.10 ± 0.732.21
C32.85 ± 1.163.53
Table 3

Real-time RT-PCR results in midgut and leg samples

Midgut samplesLeg samples
(+) Strand assay(−) Strand assay(+) Strand assay(−) Strand assay
dpiNumber (%) positive P valueaNumber (%) positive P valueStrand correlation (r)bNumber (%) positive P valueNumber (%) positive P valueStrand correlation (r)
NA, no correlation could be calculated due to < 3 positive mosquitoes. dpi, days postinfectious blood meal.
aP values, ANOVA of DENV-2 RNA copy number of positive mosquitoes samples for each day. Note: Day 0 samples were taken immediately after the blood meal.
b Pearson correlation coefficient between (+) and (−) strand DENV-2 RNA copy number.
* P ≤ 0.05.
** P ≤ 0.01.
*** P ≤ 0.001.
**** P ≤ 0.0001.
010 (100)****10 (100)****0.830 (0) NA0 (0) NANA
110 (100)****10 (100)****0.711 (10) NA1 (10) NANA
29 (90)****9 (90)****0.640 (0) NA0 (0) NANA
310 (100)****10 (100)****0.930 (0) NA0 (0) NANA
48 (80)****8 (80)****0.991 (10) NA0 (0) NANA
58 (80)****8 (80)****0.973 (30)**2 (20)NA
610 (100)****10 (100)****0.965 (50)*1 (10) NANA
710 (100)****10 (100)****0.874 (40)****2 (20)NA
89 (90)****9 (90)****1.005 (50)****3 (30)**0.87
910 (100)****10 (100)****0.959 (90)****7 (70)**0.91
109 (90)****9 (90)****0.986 (60)****6 (60)****0.90
1110 (100)****10 (100)****0.927 (70)****7 (70)****0.96
128 (80)****8 (80)****1.008 (80)****8 (80)****0.94
1310 (100)****10 (100)****0.999 (90)****9 (90)****0.95
148 (80)****8 (80)****0.998 (80)****8 (80)****0.97
Figure 1.
Figure 1.

Reproducibility of DENV-2 quantitative real-time RT-PCR assays. The CV of three CT estimates was calculated among plasmid standards analyzed on the same plate. These CVs were then compared with the CVs of CT among the same plasmid standards but analyzed on different plates on different days.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 74, 1; 10.4269/ajtmh.2006.74.132

Figure 2.
Figure 2.

Total RNA estimated with the RiboGreen assay. RNA from 10 midguts was quantified on each day. Whisker caps represent 10th and 90th percentiles. Box limits represent 25th and 75th percentiles. Lines within the boxes mark the median. Circles indicate the 5th and 95th percentiles. Day 0 corresponds to mosquitoes dissected immediately after they blood-fed.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 74, 1; 10.4269/ajtmh.2006.74.132

Figure 3.
Figure 3.

Plaque assay vs. DENV-2 RNA copies with five different DENV-2 genotypes grown in C6/36 cells. The regression equation is DENV-2 copies = 0.242 PFU + 9.09 (R2 = 0.91). Both slope and y-intercept were significantly > 0 (P ≤ 0.0001). Line shown in the lower right-hand side of the graph is the RNA copy = PFU line.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 74, 1; 10.4269/ajtmh.2006.74.132

Figure 4.
Figure 4.

Plaque assay vs. DENV-2 RNA copies in two replicate groups of D2S3 Ae. aegypti mosquitoes. The regression equations are DENV-2 copies = 0.358 PFU + 6.01 (R2 = 0.45) and DENV-2 copies = 0.335 PFU + 5.73 (R2 = 0.36).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 74, 1; 10.4269/ajtmh.2006.74.132

Figure 5.
Figure 5.

Quantitative analysis of DENV-2 replication in the midgut of individual Aedes aegypti (Chetumal strain). Y error bars represent the standard deviation of DENV-2 RNA copies in the mosquitoes testing positive on each day. Bars indicate percentage of mosquitoes with a disseminated infection. Refer to Table 3 for number of positive midguts at each time point. Day 0 corresponds to mosquitoes dissected immediately after they blood-fed.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 74, 1; 10.4269/ajtmh.2006.74.132

Figure 6.
Figure 6.

Quantitative analysis of DENV-2 replication in leg tissue from individual Aedes aegypti (Chetumal strain). Y error bars represent the standard deviation of DENV-2 RNA copies in the mosquitoes testing positive for each day. Bars indicate percentage of mosquitoes with DENV-2 (+) RNA in legs. Table 3 lists the number of positive mosquitoes at each time point.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 74, 1; 10.4269/ajtmh.2006.74.132

Figure 7.
Figure 7.

Comparison of DENV-2 (+) RNA in midguts and legs over the course of the extrinsic incubation period.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 74, 1; 10.4269/ajtmh.2006.74.132

*

Address correspondence to William Black, IV, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523. E-mail: wcb4@lamar.colostate.edu

Authors’ addresses: Jason Richardson, Alvaro Molina-Cruz, Ma Isabel Salazar, and William Black, IV, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, E-mails: jasonr@calostate.edu and web4@lamar.colostate.edu.

Acknowledgments: The authors thank the staff and students at AIDL for their assistance and helpful suggestions, especially Dr. Francisco Díaz.

Financial support: Funding for this research was provided from National Institutes of Health, Grants No. R01AI49256 and No. U01AI45430.

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Author Notes

Reprint requests: William Black, IV, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, Telephone: (970) 491-8530, Fax: 970-491-1815, E-mail: web4@lamar.colostate.edu.
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