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    Inhibition of Ae. aegypti midgut trypsin activity by STI. Midgut late trypsin mRNA levels by qRT-PCR (A), midgut late trypsin protein by Western blot (B), ovary development (C, D), and oviposition (E) after a blood meal with (+) or without (−) STI. A second uninfected blood feeding (2BF) without STI was done 7 days after the infectious blood feeding. Error bars represent standard error; different letters are significantly different (ANOVA, P < 0.05). qRT-PCR data was normalized with ribosomal S6 mRNA levels. Ovary images were taken at the same magnification. The number of mosquitoes (n) is indicated in E.

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    Effect of inhibiting Ae. aegypti midgut trypsins with STI, during digestion of a DENV-2–infected blood meal, on the viral infection of the midgut. Midgut DENV-2 (+)RNA by qRT-PCR (A, C, D), Western blot of midgut DENV-2 E protein (B, E), and DENV-2 (−)RNA by qRT-PCR (C) after a DENV-2–infected blood meal with (+) or without (−) STI. A second uninfected blood feeding (2BF) without STI was done 7 days after the infectious blood feeding. Error bars represent standard error; different letters within a day are significantly different (two-way ANOVA, P < 0.05). qRT-PCR data was normalized with ribosomal S6 mRNA levels.

  • View in gallery

    Effect of inhibiting Ae. aegypti midgut trypsins with STI, during digestion of a DENV-2–infected blood meal, on the dissemination of the virus from the midgut. DENV-2 (−)RNA by qRT-PCR in thorax (A), IFA of DENV-2 E protein on mosquito heads by Day 14 postinfection (B) with a DENV-2–infected blood meal with (+) or without (−) STI (-STI N = 45; +STI N = 20). qRT-PCR data was normalized with ribosomal S6 mRNA levels. Head infection levels were uninfected (−) and infected in four increasing levels (+, ++, +++, ++++). Error bars represent standard error. Frequency distributions for head infections with and without STI were significantly different (χ2, P < 0.05).

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    Effect of bovine trypsin in vitro digestion of DENV-2 culture on infection of Ae. aegypti midguts. DENV-2 (+)RNA by qRT-PCR (A) and DENV-2 E protein Western blot (B) were detected on midguts. In vitro digestion with bovine trypsin was done on DENV-2 culture (V) or cell culture control (C) and was stopped with STI. Error bars represent standard error; different letters within a day are significantly different (two-way ANOVA, P < 0.05). qRT-PCR data was normalized with ribosomal S6 mRNA levels.

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EFFECT OF MOSQUITO MIDGUT TRYPSIN ACTIVITY ON DENGUE-2 VIRUS INFECTION AND DISSEMINATION IN AEDES AEGYPTI

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  • 1 Department of Microbiology, Immunology and Pathology, and the Arthropod-borne and Infectious Diseases Laboratory, Colorado State University, Fort Collins, Colorado; Laboratory of Malaria and Vector Research, Medical Entomology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland

The effect of mosquito midgut trypsins in dengue serotype 2 flavivirus (DENV-2) infectivity to Aedes aegypti was studied. Addition of soybean trypsin inhibitor (STI) in a DENV-2 infectious blood meal resulted in a 91–97% decrease in midgut DENV-2 RNA copies (qRT-PCR analysis). STI treatment also resulted in slower DENV-2 replication in the midgut, less DENV-2 E protein expression, and decreased dissemination to the thorax and the head. A second uninfected blood meal, 7 days after the STI-treated infectious meal, significantly increased DENV-2 replication in the midgut and recovered oogenesis, suggesting that the lower viral infection caused by STI was in part due to a nutritional effect. Mosquitoes fed DENV-2 digested in vitro with bovine trypsin (before STI addition) exhibited a transient increase in midgut DENV-2 4 days postinfection. Blood digestion and possibly DENV-2 proteolytic processing, mediated by midgut trypsins, influence the rate of DENV-2 infection, replication, and dissemination in Ae. aegypti.

INTRODUCTION

Aedes aegypti is a container-breeding mosquito with a cosmopolitan distribution in tropical and subtropical regions of the world and on a global basis is a common vector of yellow fever and dengue fever flaviviruses (DENV).1 Since the demise of mosquito-control programs beginning in the late 1960s, Ae. aegypti reestablished itself throughout tropical and subtropical areas of the Americas. Despite the widespread availability of an effective and safe vaccine, yellow fever remains an important public health problem in much of Africa and South America.2 Aedes aegypti is also the most prevalent vector of DENV (serotypes 1–4) in a human-mosquito cycle. Dengue fever is one of the most rapidly expanding diseases in the tropics, with more than 2 billion people at risk. All four serotypes of the virus are now circulating in the Americas, and an estimated 100 million human infections occur annually.

Vector competence refers to the intrinsic permissiveness of an arthropod vector to infection, replication, and transmission of a virus.3,4 The midgut is the first organ that an arbovirus encounters; it can prevent the invasion and replication of the viruses (midgut infection barrier) or the dissemination to other tissues (midgut escape barrier). The molecular nature of these barriers is unknown, but they have been found to be major determinants of vector competence to DENV during experimental infections. The barriers also vary in prevalence in natural populations, leading to large intraspecific variation of Ae. aegypti vector competence to DENV.57

DENV are (+)RNA viruses that for replication synthesize a complementary (−)RNA.8 Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) has been shown to be a rapid and sensitive method to quantify DENV.915

The early events of flavivirus midgut infection are not well understood. They presumably involve receptor binding and some midgut cell penetration mechanism. DENV have been shown to penetrate C6/36 mosquito cells in culture by membrane fusion and sometimes by receptor-mediated endocytosis.16,17 Other arboviruses (bunya- and orbiviruses) have been shown to increase affinity for the midgut after proteolytic processing of virion surface proteins.1821 With this precedent, we decided to test the involvement of midgut trypsins in determining DENV infectivity in Ae. aegypti.

Midgut trypsins play a central role during blood digestion in Aedes aegypti.22 This mosquito synthesizes early trypsin and late trypsin proteins de novo upon blood feeding. Early trypsin activity peaks 3 hours after blood feeding and then drops within a few hours. Early trypsin activity regulates late trypsin mRNA synthesis, which reaches a maximum level 24 hours after feeding, followed by an increase in late trypsin protein, which reaches 4–6 μg/midgut. Late trypsin accounts for most of the endoproteolytic activity during blood digestion in the Ae. aegypti midgut.22 Addition of an excess of soybean trypsin inhibitor (STI) in the blood meal inhibits blood digestion23 by inhibiting early trypsin activity and the expression of late trypsin.24 We evaluated the effect of inhibiting mosquito midgut trypsins with STI on DENV infectivity to Ae. aegypti during the digestion of a DENV-2–infected blood meal. We also assessed whether in vitro digestion of DENV-2 in cell culture with bovine trypsin recovers infectivity to STI-treated mosquitoes.

MATERIALS AND METHODS

Aedes aegypti rearing.

Mosquitoes used were the F5 generation of a wild population from Chetumal, Mexico. This population is highly susceptible to DENV-2.5 Eggs were hatched and mosquitoes were raised at a constant temperature of 27°C and 80% relative humidity in an insectary with a 12:12 hour photoperiod. Eggs were collected in water cups containing paper filters.

DENV-2 mosquito infections.

The Jamaica 1409 strain of DENV-225 was amplified in C6/36 cells.5,26 Briefly, 0.5 mL aliquots of virus stock were used to infect 75 cm2 flasks of confluent C6/36 cells at a multiplicity of infection of 1.5 virus particles/cell. Infected cells were incubated for 14 days at 28°C in L15 medium supplemented with 2% heat-inactivated fetal bovine serum, penicillin (1%), streptomycin (1%), and l-glutamine (1%). Medium was changed on Day 7. Virus and cells were harvested on Day 14 with a cell scraper. The virus suspension was mixed 1:1 with defibrinated sheep blood. ATP was added to a final concentration of 1 mM, STI was added to a final concentration of 2 mg/mL as previously used,24 and the blood meal was incubated at 38°C for 15 minutes. The blood meal was placed in membrane feeders covered with hog gut. Blood meal virus titers were determined by inoculating serial 10-fold dilutions of the respective meal onto C6/36 cells in the wells of a 96-well plate. Cells were assayed by immunofluoresence, and tissue culture infectious dose 50% (TCID50) titers were determined.26 The final infectious blood meal virus titer had a 109.5 TCID50/mL in the sample without STI and 109.2 TCID50/mL in the sample with STI.

For mosquito infections, 400–500 mosquitoes/4-L carton were starved of sucrose and deprived of water for 36 hours prior to blood feeding. Blood meals were maintained at a constant temperature of 37°C. Mosquitoes that were 3–4 days old were allowed to feed for 45–60 minutes. Fully engorged mosquitoes were selected and held in the insectary. Untreated control mosquitoes were provided with 10% protease-free sucrose (Sigma, St. Louis, MO) while the sucrose in the STI-treated mosquitoes was laced with 2 mg/mL STI. Samples of 20 mosquitoes were collected 1, 2, 4, 6, 7, 11, and 14 days after feeding. Mosquitoes were frozen in dry-ice ethanol and kept at −70°C awaiting further analysis.

In vitro digestion with bovine trypsin.

One volume of 14-day DENV-2 culture in C6/36 cells was digested with 0.1 mg/mL bovine trypsin (12,700 units/mg, Sigma T-1426) for 15 minutes with shaking at room temperature. The reaction was stopped by adding STI to a final concentration of 8 mg/mL and incubating for another 15 minutes with shaking at room temperature. Afterwards, the digested viral culture was diluted with 1 volume of 14-day-old C6/36 and 2 volumes of defibrinated sheep blood (Treatment 3; Table 1). To assess the potential effects of cell protein digestion in the viral culture, a digested cell control (Treatment 2; Table 1) was prepared by predigesting with trypsin uninfected C6/36 cells, stopping the reaction with STI, and diluting with 1 volume of 14-day-old cells and 2 volumes of blood. The undigested control (Treatment 1; Table 1) was prepared with 1 volume of DENV-2 culture, 1 volume of uninfected C6/36 cell culture, and 2 volumes of blood. The infectious blood meal virus titer was 109.2 TCID50/mL for the undigested control and 108.8 TCID50/mL for both the in vitro trypsin digested samples.

Mosquito dissection and RNA extraction.

Mosquito mid-guts, thoraces, and ovaries were dissected in 25 μL of RNAlater (Sigma). Total RNA was extracted in duplicate using the RNAeasy kit (Qiagen, Valencia, CA) from pools of five midguts or five thoraces. Total RNA was eluted in 50 μL of RNAase-free water. Ovaries were fixed in 4% paraformaldehyde in PBS and examined by confocal microscopy.

Late trypsin qRT-PCR.

Late trypsin mRNA was measured 24 hours after the blood meal by SYBR green qRT-PCR (Qiagen). Primers were F1 (5′-ACAGTACCAGTATTCG-GCAAA-3′) and R1 (5′-GAGAACTTGGAATGG-GAACT-3′). The target fragment (147 bp) was TA cloned into a pCR2.1 (Invitrogen, Carlsbad, CA) and used to generate a standard curve with 101–108 copies/reaction. Reactions (20 μL) with 4 μL of total RNA were carried out in an Opticon-2 system (MJ Research, Reno, NV). cDNA was synthesized at 50°C for 30 minutes, reverse transcriptase was inactivated at 95°C for 15 minutes, PCR involved 44 cycles of 15 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C. Fluorescence readings were taken at 72°C after each cycle. A final extension at 72°C for 5 minutes was completed before deriving a melting curve (70–95°C) to confirm the identity of the PCR product. qRT-PCR measurements were made in triplicate. Results were normalized with ribosomal protein S6 (RPS6) as an internal standard, also measured by qRT-PCR, and expressed in copies/midgut.

DENV-2 RNA qRT-PCR.

DENV-2 (+)RNA was measured by qRT-PCR as above using primers For (5′-ACAAGTCGAACAACCTGGTCCAT-3′) and Rev (5′-GCCGCACCATTGGTCTTCTC-3′) directed to the NS5 gene.27 A standard curve was generated by analyzing 101 to 108 copies/reaction of plasmid containing the 177-bp target fragment. Results were normalized with RPS6 and expressed in copies/midgut. DENV-2 (−)RNA was measured by adding the For primer, denaturing RNA at 80°C for 5 minutes before the cDNA synthesis, and the second primer (Rev) was added only after inactivation of the reverse transcriptase for 15 minutes at 95°C. Controls lacking both primers during the cDNA synthesis were done to confirm reverse transcriptase inactivation.

RPS6 qRT-PCR.

RPS6 mRNA was measured by qRT-PCR as above using primers RPS6(5+) (5′-CGTCGTCA-GGAACGTATCCG3-′) and RPS6(5−) (5′-TCTTG-GCAGCCTTAGCAGC-3′). A standard curve was generated analyzing 101 to 108 copies/reaction of plasmid containing the 118-bp fragment. Results were expressed in copies/midgut.

DENV-2 IFA.

DENV-2 was detected by indirect immunofluoresence in head squashes.5 A mouse-derived monoclonal antibody 3H5,28 directed against a flavivirus E protein epitope, was the primary antibody.

Late trypsin and DENV-2 Western blots.

Aedes aegypti midguts infected with DENV-2 were dissected and proteins were extracted in pools of five midguts by homogenizing them with 50 μL of 1X PBS containing protease inhibitors. Supernatant was collected after centrifugation (12,000 rpm for10 minutes). To denature the proteins, supernatants were mixed with 1 volume of 2X SDS sample buffer and boiled for 5 minutes. The equivalent of one midgut/lane (20 μL) was run in a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The protein was transferred to a polyvinylidine difluoride (PVDF) membrane using the Transblot system (Bio-Rad, Hercules, CA). Western blot for DENV-2 was done with mouse monoclonal antibody 1A6A-8 against E-glycoprotein,29 and late trypsin was detected with a specific mouse monoclonal antibody.30 First antibody was detected with an anti-mouse/phosphatase conjugate.

RESULTS

Inhibition of midgut trypsin activity and blood digestion by STI.

Late trypsin accounts for most of the endoproteolytic activity during blood digestion in the Ae. aegypti midgut, and its expression is regulated by the activity of early trypsin.22 Mosquitoes were membrane fed a DENV-2–infected blood meal with or without 2 mg/mL STI. The extent of midgut trypsin inhibition by STI was assessed by measuring late trypsin mRNA and protein levels after blood feeding. Minimal amounts of late trypsin mRNA were detected by qRT-PCR in midguts from STI-treated mosquitoes 24 hours after blood feeding. These were not significantly different from prefeeding levels and were 97% lower than in untreated individuals (Figure 1A). Late trypsin Western blot analysis on midguts was consistent with mRNA data. Midguts from STI-treated mosquitoes showed minimal amounts of late trypsin protein 24 hours after blood feeding compared with untreated, and no late trypsin protein was detected 48 hours after blood feeding (Figure 1B). Both observations indicate that midgut trypsin activity was substantially inhibited by STI.

The level of inhibition of blood digestion by STI was determined by assessing oogenesis because this correlates with the amount of blood digested by the mosquito.23 Whereas untreated mosquitoes presented normal egg development by 48 hours after blood feeding (Figure 1C), most STI-treated mosquitoes had no detectable egg development (Figure 1D). Consistent with these results, STI-treated mosquitoes produced only 1% of the eggs laid by untreated mosquitoes (Figure 1E). Mosquitoes treated with STI remained engorged for an average of 3 days after blood feeding and then excreted the blood meal. The almost complete inhibition of oogenesis by STI indicates that the inhibition of trypsin activity nearly completely inhibited blood digestion.

The reversibility of the inhibition of digestion by STI was tested by providing mosquitoes with a second, untreated blood meal 7 days after receiving the infectious blood meal with STI. Administration of a second blood meal resulted in higher levels of oviposition in the group that had received STI in their first meal (Figure 1E). This shows that the initial lack of oviposition was due to the nutritional effect of STI and that it had not damaged the ovaries. No significant differences in mortality 7 days postinfection was observed between STI treated (8.6% ± 9) or untreated mosquitoes (7.1% ± 5.8), and STI had no observable toxic effects.

Effect of STI on DENV-2 midgut infection.

STI-treated mosquito midguts developed a DENV-2 infection but at much lower titers and with slower replication of the virus when compared with midguts from untreated mosquitoes. The levels of DENV-2 (+)RNA from Days 4 to 7 in midguts from STI-treated mosquitoes were 91–97% lower than in the untreated group (Figure 2A). In the case of viral protein, midguts from STI-treated mosquitoes did not have detectable DENV-2 E protein up to Day 14 after blood feeding, whereas in midguts from untreated mosquitoes it was detectable at Day 4 (Figures 2B and 2E). The amount of DENV-2 (-)RNA was also measured to evaluate replication intermediates as indicators of active infections.3134 Midguts from STI-treated mosquitoes also had lower levels of DENV-2 (-)RNA from Days 4 to 11 when compared with untreated mosquitoes (Figure 2C). Although the amount of DENV-2 (−)RNA was 102– 103 lower, the courses of infection as detected by DENV-2 (−)RNA and DENV-2 (+)RNA followed a similar pattern (Figure 2C).

We also tested the hypothesis that STI lowers DENV-2 infection because of lack of nutrients. Mosquitoes received a second uninfected blood meal with no STI 7 days after the infectious one. When given a second blood meal, the levels of DENV-2 (+)RNA measured in midguts of STI-treated mosquitoes were not significantly different from those of untreated ones (Figure 2D) at Days 11 and 14. The second blood meal also recovered viral E protein expression by Day 14, not by Day 11, as detected by Western blot (Figure 2E). These results suggest that the lower DENV-2 midgut infection with STI is in part caused by the lack of nutrients, most probably amino acids, normally provided by blood digestion.

DENV-2 dissemination.

The effect of inhibiting midgut trypsin activity on DENV-2 dissemination from the midgut was studied by measuring DENV-2 (−)RNA in the thorax, which is direct evidence of DEN replication in a tissue and decreases the possibility of measuring (+)RNA from contaminating viral particles released by the midgut during dissection. No significant viral dissemination was detected before Day 7. Mosquitoes that received STI in the infectious blood meal showed lower amounts of DENV-2 (−)RNA in the thorax by Days 11 and 14 postinfection than untreated mosquitoes (Figure 3A). Similar results were obtained by immuno fluorescent assay (IFA) on heads. Only 16% of untreated mosquitoes were negative for DENV-2 E protein in the head by Day 14 as compared with 35% of STI-treated ones (Figure 3B). STI treatment also led to a lower infection titer in the head (Figure 3B). These results indicate that midgut trypsin activity also affects the rate of dissemination from the midgut and could therefore constitute a midgut escape barrier.

In vitro digestion with bovine trypsin.

Mosquitoes were fed a meal with a 50% volume of sheep blood, 25% uninfected cell culture, and 25% DENV-2–infected culture. Three different treatments were compared (see Table 1). The infectious blood meal virus titer was 109.2 TCID50/mL for the undigested control and 108.8 TCID50/mL for both the in vitro trypsin-digested samples. At Day 4 postinfection, trysin pre-digestion of the viral culture rescued the level of DENV-2 (+)RNA in the midgut to levels even higher than in mosquitoes fed a meal without STI or one in which uninfected cell culture was predigested (Figure 4A). This result indicates that direct contact of trypsin with the virus is necessary to enhance the infectivity. This enhancement was confirmed by Western blot analysis, where in spite of the presence of STI in the meal, DENV-2 E protein was clearly detected (Figure 4B). Interestingly, the observed enhancement was transient, as by day 6 postinfection there was no difference in DENV-2 infectivity between midguts with the meals in which the uninfected or the DENV-2–infected culture were predigested with trypsin (Figure 4A), and the E protein could not be detected in either sample (Figure 4B). Mosquitoes that fed on DENV-2 culture or trypsin-treated uninfected cell culture that were then inhibited with STI had large reductions in oviposition rates (96% and 95.5%, respectively) as compared with the untreated control (84.75 eggs/female, N = 94).

DISCUSSION

The use of qRT-PCR to determine DENV-2 RNA levels in specific mosquito tissues allowed a fast and detailed analysis of the time course of DENV-2 infection. After the administration of a regular infectious blood meal, not containing STI, there was a decrease in midgut DENV-2 titer on the second day postinfection (Figure 2C). This decrease corresponds to the eclipse phase due to elimination of the virus present in the blood meal,3,4 and it was followed by a steady increase in DENV-2 RNA up to 7 days postinfection (Figure 2A). This time course of midgut infection was similar to patterns of infections of Culex tarsalis35 and Culex pipiens midgut36 with Western equine encephalitis virus and Japanese B encephalitis virus, respectively. At Day 11, however, a decline in DENV-2 RNA levels was observed (Figure 2A), which could be due in part to a general reduction in midgut metabolic activity. Alternatively, one cannot dismiss the possibility that the midgut could be actively eliminating the virus. Administration of a second noninfectious meal did not significantly increase DENV-2 RNA levels at Days 11 and 14 (Figure 2D), indicating that midgut viral RNA levels reached at this time are not influenced by nutrient availability. In contrast, the second meal apparently resulted in a pronounced increase in E protein expression at Day 14 (Figure 2E), indicating that the availability of dietary amino acids could be limiting protein translation. By the time midgut DENV-2 RNA levels begin to decline (Day 11), the virus can be detected in the thorax and increases dramatically in this tissue by Day 14 postinfection (Figure 3A). At Day 14, the virus has also disseminated to the head, as 84% of the females were positive for DENV-2 by immunofluorescence (Figure 3B).

Inhibition of midgut trypsin activity during an infectious blood feeding led to a significantly lower titer of DENV-2 in the midgut and to a lower rate of DENV-2 dissemination (Figures 2 and 3). Midgut trypsin activity appears to be required for DENV-2 to achieve optimal levels of infection, replication, and dissemination in the midgut. Inhibition appears to be due in part to a nutritional effect, as a second uninfected blood meal recovered DENV-2 mRNA titers of STI-treated mosquitoes (at Days 11 and 14) and protein E translation (at Day 14) back to control levels (Figure 2D).

The Ae. aegypti midgut undergoes major changes in morphology and gene expression after blood feeding. These changes include dispersion of mitochondria and rearrangement of the rough endoplasmic reticulum, suggesting large-scale metabolic shifts in the blood-fed midgut.37,38 Blood feeding also triggers changes in expression of at least 333 midgut genes, including upregulation of genes involved in protein and amino acid metabolism, peritrophic matrix-formation, and iron metabolism.39 Our results suggest that the metabolic changes in the midgut elicited by blood meal digestion are required for optimal DENV-2 replication and dissemination. In STI-treated mosquitoes, midgut DENV-2 mRNA titers increased slowly over time (Figure 2C), but E protein could not be detected up to 14 days postfeeding, indicating that the nutritional effect was more dramatic at the level of protein translation. The inhibition of blood digestion presumably prevented the establishment of an adequate mid-gut amino acid supply to support the synthesis of detectable amounts of DENV-2 E protein in the midgut. This nutritional deficit did not affect other tissues to the same extent as the midgut, as IFA detected E protein in the heads of 65% of STI-treated mosquitoes (although at lower levels than the control group) by Day 14 (Figure 3B). The lack of detection of DENV-2 E protein by Western blot in the midgut of STI-treated mosquitoes up to Day 14 suggests lack of sensitivity of this method. DENV-2 E protein must have been produced in the midgut in order for the virus to disseminate.

The increase in DENV-2 infection by blood meal ingestion could have epidemiologic implications. Ingestion of multiple uninfected blood feedings may boost a previous incipient infection. This seems plausible, as Ae. aegypti tends to take small sequential blood-feeds on humans.40 Our results show that even very low levels of virus can remain “dormant” in the midgut for a week (and probably longer) and then be reactivated by ingestion of uninfected blood, generating high infection titers.

The fact that the level of trypsin activity affects DENV-2 midgut infection and dissemination suggests that natural variations in the rate of induction, or absolute expression levels of midgut trypsins, could result in midgut infection and dissemination barriers. Natural variation in midgut trypsin activity could be genetically or environmentally determined. Consistent with this hypothesis, quantitative trait loci (QTL) analysis of a genetic cross between mosquitoes susceptible and refractory to dengue infection revealed that the disseminated infection rate correlated positively and additively with the number of susceptible alleles present for both early trypsin (Chr. II) and late trypsin (Chr. III) genes.41 It is also known that environmental temperature affects both the rate of enzyme synthesis and activity in Ae. aegypti.42,43 Blood digestion in Ae. aegypti at 32°C is twice as fast compared with 22°C.42

In vitro digestion of the viral culture (in C6/36 cells) prior to the addition of STI to the blood meal increased the amount of DENV-2 mRNA and E protein in the Ae. aegypti midgut by Day 4 (Figures 4A and 4B) but not by Day 6. This suggests that tryptic digestion of viral surface proteins enhanced the interaction of DENV-2 with the midgut cells during the first days postinfection but was unable to support viral replication. It is unlikely that the transient increase in infectivity associated with trypsin predigestion of the viral culture was due to a nutritional effect, as this phenomenon was not observed in the control group in which the uninfected cell culture was predigested instead of the DENV-2–infected culture. Direct contact of the protease with the virus was required to enhance its association with the midgut. Furthermore, in vitro pre-digestion of the meal (infected or uninfected) had little nutritional value, as only minimal egg production was observed in all STI-treated samples.

Enhancement of arbovirus infectivity in the insect midgut by proteolytic processing has previously been shown for La Crosse virus (Bunyavirus) in Ochlerotatus triseriatus18,19 or for Bluetongue virus (Orbivirus) in Culicoides biting midges.20,21 In those cases, proteolytic processing of viral glycoprotein increased binding to midgut cells incubated in vitro. A similar mechanism could underlie the effect of trypsin on DENV-2 in Ae. aegypti we have observed in vivo. It has been shown previously that cleavage of prM protein into pr and M proteins is associated with an increase of infectivity of dengue viruses in Vero cells44 and of tick-borne encephalitis virus (Flavivirus) in C6/36,45 procine,46,47 and in BHK-21 cells.48 Cleavage of prM could be involved in the enhanced infectivity of DENV-2 we observed, as trypsin has been shown to be capable of processing prM protein in tick-borne encephalitis virus.48 Further experiments are underway to elucidate the subcellular localization of DENV-2 during this period of enhanced interaction and the mechanism(s), such as modification of specific viral surface proteins, which could mediate this phenomenon.

In summary, we have shown that midgut trypsin activity affects DENV-2 infection and dissemination in the Ae. aegypti midgut. Midgut trypsin activity facilitates DEN infection in Ae. aegypti through a nutritional effect and probably also by direct proteolytic processing of the viral surface.

Table 1

Treatments used to test for the effects of in vitro digestion with bovine trypsin*

* STI = soybean trypsin inhibitor.
  1. Undigested control

    1 vol. Viral culture plus 1 vol. Cell culture plus 2 vol. blood

  2. In vitro trypsin pre-digested cell culture control

        1 vol. Cell culture →15 min Bovine Trypsin →15 min STI →add 1 vol. Viral culture and 2 vol blood

  3. In vitro trypsin pre-digested viral culture

        1 vol. Viral culture →15 min Bovine Trypsin →15 min STI →add 1 vol. Cell culture and 2 vol. blood

Figure 1.
Figure 1.

Inhibition of Ae. aegypti midgut trypsin activity by STI. Midgut late trypsin mRNA levels by qRT-PCR (A), midgut late trypsin protein by Western blot (B), ovary development (C, D), and oviposition (E) after a blood meal with (+) or without (−) STI. A second uninfected blood feeding (2BF) without STI was done 7 days after the infectious blood feeding. Error bars represent standard error; different letters are significantly different (ANOVA, P < 0.05). qRT-PCR data was normalized with ribosomal S6 mRNA levels. Ovary images were taken at the same magnification. The number of mosquitoes (n) is indicated in E.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 5; 10.4269/ajtmh.2005.72.631

Figure 2.
Figure 2.

Effect of inhibiting Ae. aegypti midgut trypsins with STI, during digestion of a DENV-2–infected blood meal, on the viral infection of the midgut. Midgut DENV-2 (+)RNA by qRT-PCR (A, C, D), Western blot of midgut DENV-2 E protein (B, E), and DENV-2 (−)RNA by qRT-PCR (C) after a DENV-2–infected blood meal with (+) or without (−) STI. A second uninfected blood feeding (2BF) without STI was done 7 days after the infectious blood feeding. Error bars represent standard error; different letters within a day are significantly different (two-way ANOVA, P < 0.05). qRT-PCR data was normalized with ribosomal S6 mRNA levels.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 5; 10.4269/ajtmh.2005.72.631

Figure 3.
Figure 3.

Effect of inhibiting Ae. aegypti midgut trypsins with STI, during digestion of a DENV-2–infected blood meal, on the dissemination of the virus from the midgut. DENV-2 (−)RNA by qRT-PCR in thorax (A), IFA of DENV-2 E protein on mosquito heads by Day 14 postinfection (B) with a DENV-2–infected blood meal with (+) or without (−) STI (-STI N = 45; +STI N = 20). qRT-PCR data was normalized with ribosomal S6 mRNA levels. Head infection levels were uninfected (−) and infected in four increasing levels (+, ++, +++, ++++). Error bars represent standard error. Frequency distributions for head infections with and without STI were significantly different (χ2, P < 0.05).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 5; 10.4269/ajtmh.2005.72.631

Figure 4.
Figure 4.

Effect of bovine trypsin in vitro digestion of DENV-2 culture on infection of Ae. aegypti midguts. DENV-2 (+)RNA by qRT-PCR (A) and DENV-2 E protein Western blot (B) were detected on midguts. In vitro digestion with bovine trypsin was done on DENV-2 culture (V) or cell culture control (C) and was stopped with STI. Error bars represent standard error; different letters within a day are significantly different (two-way ANOVA, P < 0.05). qRT-PCR data was normalized with ribosomal S6 mRNA levels.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 5; 10.4269/ajtmh.2005.72.631

Authors’ addresses: Alvaro Molina-Cruz (E-mail: amolina-cruz@niaid.nih.gov), Lalita Gupta (E-mail: lgupta@niaid.nih.gov), and Carolina Barillas-Mury (E-mail: cbarillas@niaid.nih.gov), Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook III, MSC 8130, 12735 Twinbrook Parkway, Bethesda, MD 20892-8130, Telephone: (301) 435-2706, Fax: (301) 480-1337. Jason Richardson, Kristine Bennett, and William Black IV (E-mail: wcb4@colostate.edu), Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, Telephone: (970) 491-6136, Fax: 970-4911815.

Acknowledgments: Funding for this research was provided from the National Institutes of Health, grant nos. R01 AI49256, R01AI45573, and U01AI45430. The authors thank the staff and students at AIDL for their assistance and helpful suggestions, especially Dr. Francisco Díaz and Dr. Irma Sánchez-Vargas.

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