- INTRODUCTION
- MATERIALS AND METHODS
- Aedes aegypti rearing.
- Dengue virus Type 2 production.
- RNA extraction and cDNA synthesis
- Double stranded RNA (dsRNA) synthesis.
- dsRNA injection and artificial bloodfeeds.
- Quantitative reverse transcriptase polymerase chain reactions (Q-RT-PCR).
- Enzymatic activity assays.
- Trypsin inhibitors.
- Indirect immunofluorescence assays (IFA).
- Statistical analysis.
- RESULTS
- DISCUSSION
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1
Gubler DJ, 1996. The global resurgence of arboviral diseases. Trans R Soc Trop Med Hyg 90 :449–451.
-
2
Monath T, 1994. Dengue: the risk to developed and developing countries. Proc Natl Acad Sci USA 91 :2395–2400.
-
3
Nelson M, 1986. Aedes aegypti: Biology and Ecology. Washington DC: Pan American Health Organization, 1–59.
-
4
Tinker M, 1964. Larval habitat of Aedes aegypti (L.) in the United States. Mosq News 24 :426–432.
-
5
Felix CR, Betschart B, Billingsley PF, Freyvogel TA, 1991. Post-feeding induction of trypsin in the midgut of Aedes aegypti L (Diptera, Culicidae) is separable into 2 cellular phases. Insect Biochem 21 :197–203.
-
6
Noriega FG, Wang XY, Pennington JE, Barillas-Mury CV, Wells MA, 1996. Early trypsin, a female-specific midgut protease in Aedes aegypti: isolation, amino-terminal sequence determination, and cloning and sequencing of the gene. Insect Biochem Mol Biol 26 :119–126.
-
7
Pennington JE, Noriega FG, Wells MA, 1995. The expression of early trypsin in Aedes aegypti. J Cell Biochem 21A (Suppl.):211.
-
8
Graf R, Binz H, Briegel H, 1988. Monoclonal antibodies as probes for Aedes aegypti trypsin. Insect Biochem 18 :463–470.
-
9
Graf R, Raikhel AS, Brown MR, Lea AO, Briegel H, 1986. Mosquito trypsin immunocytochemical localization in the midgut of bloodfed Aedes aegypti (L). Cell Tissue Res 245 :19–27.
-
10
Barillas-Mury C, Graf R, Hagedorn HH, Wells MA, 1991. cDNA and deduced amino acid sequence of a blood meal-induced trypsin from the mosquito, Aedes aegypti. Insect Biochem 21 :825–831.
-
11
Kalhok SE, Tabak LM, Prosser DE, Brook W, Downe AE, White BN, 1993. Isolation, sequencing and characterization of two cDNA clones coding for trypsin-like enzymes from the midgut of Aedes aegypti. Insect Mol Biol 2 :71–79.
-
12
Jiang QJ, Hall M, Noriega FG, Wells M, 1997. cDNA cloning and pattern of expression of an adult, female specific chymotrypsin from Aedes aegypti midgut. Insect Biochem Mol Biol 27 :283–289.
-
13
Black WC, Bennett KE, Gorrochotegui-Escalante N, Barillas-Mury CV, Fernandez-Salas I, Munoz MD, Farfan-Ale JA, Olson KE, Beaty BJ, 2002. Flavivirus susceptibility in Aedes aegypti. Arch Med Res 33 :379–388.
-
14
Noriega FG, Wells MA, 1999. A molecular view of trypsin synthesis in the midgut of Aedes aegypti. J Insect Physiol 45 :613–620.
-
15
Pennington J, Wells M, 2005. The adult midgut: structure and function. Marquardt W, Black WC IV, Freier J, Hagerdorn H, Hemingway J, Higgs S, James A, Kondratieff B, Moore C, eds. Biology of Disease Vectors. Burlington, MA: Elsevier Academic Press, 289–295.
-
16
Espejo RT, Lopez S, Arias C, 1981. Structural polypeptides of simian rotavirus Sa11 and the effect of trypsin. J Virol 37 :156–160.
-
17
Estes MK, Graham DY, Mason BB, 1981. Proteolytic enhancement of rotavirus infectivity—molecular mechanisms. J Virol 39 :879–888.
-
18
Ludwig GV, Christensen BM, Yuill TM, Schultz KT, 1989. Enzyme processing of La Crosse virus glycoprotein G1: a bunyavirus-vector infection model. Virology 171 :108–113.
-
19
Mertens PP, Burroughs JN, Walton A, Wellby MP, Fu H, OHara ‘ RS, Brookes SM, Mellor PS, 1996. Enhanced infectivity of modified bluetongue virus particles for two insect cell lines and for two Culicoides vector species. Virology 217 :582–593.
-
20
Bennett KE, Flick D, Fleming KH, Jochim R, Beaty BJ, Black WC IV, 2005. Quantitative trait loci that control dengue-2 virus dissemination in the mosquito Aedes aegypti. Genetics 170 :185–194.
-
21
Bosio CF, Fulton RE, Salasek ML, Beaty BJ, Black WC IV, 2000. Quantitative trait loci that control vector competence for dengue-2 virus in the mosquito Aedes aegypti. Genetics 156 :687–698.
-
22
Molina-Cruz A, Gupta L, Richardson J, Bennett K, Black WC IV, Barillas-Mury C, 2005. Effect of mosquito midgut trypsin activity on dengue-2 virus and dissemination in Aedes aegypti. Am J Trop Med Hyg 72 :631–637.
-
23
Pierro DJ, Salazar MI, Beaty BJ, Olson KE, 2006. Infectious clone construction of dengue virus type 2, strain Jamaican 1409, and characterization of a conditional E6 mutation. J Gen Virol 87 :2263–2268.
-
24
Keene KM, Foy BD, Sanchez-Vargas I, Beaty BJ, Blair CD, Olson KE, 2004. RNA interference acts as a natural antiviral response to O’nyong-nyong virus (Alphavirus; Togaviridae) infection of Anopheles gambiae. Proc Natl Acad Sci USA 101 :17240–17245.
-
25
Bennett K, Olson K, Munoz Mde L, Fernandez-Salas I, Farfan-Ale J, Higgs S, Black WC IV, Beaty B, 2002. Variation in vector competence for dengue 2 virus among 24 collections of Aedes aegypti from Mexico and the United States. Am J Trop Med Hyg 67 :85–92.
-
26
Lu SJ, Pennington JE, Stonehouse AR, Mobula MM, Wells MA, 2006. Reevaluation of the role of early trypsin activity in the transcriptional activation of the late trypsin gene in the mosquito Aedes aegypti. Insect Biochem Mol Biol 36 :336–343.
-
27
Houk EJ, Kramer LD, Hardy JL, Chiles RE, 1985. Western equine encephalomyelitis virus - In vivo infection and morphogenesis in mosquito mesenteronal epithelial cells. Virus Res 2 :123–138.
-
28
Whitfield SG, Murphy FA, Sudia WD, 1973. St. Louis encephalitis virus: An ultrastructural study of infection in a mosquito vector. Virology 56 :70–87.
-
29
Brown SE, Severson DW, Smith LA, Knudson DL, 2001. Integration of the Aedes aegypti mosquito genetic linkage and physical maps. Genetics 157 :1299–1305.
-
30
Hacker JK, Volkman LE, Hardy JL, 1995. Requirement for the G1-protein of California encephalitis virus in infection in vitro and in vivo. Virology 206 :945–953.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 260 | 215 | 16 |
| Full Text Views | 280 | 9 | 0 |
| PDF Downloads | 109 | 10 | 0 |
The Effects of Midgut Serine Proteases on Dengue Virus Type 2 Infectivity of Aedes aegypti
Doug E. Brackney
Doug E. Brackney
Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado
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Brian D. Foy
Brian D. Foy
Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado
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Ken E. Olson
Ken E. Olson
Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado
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Dengue viruses (DENV) cause significant morbidity and mortality worldwide and are transmitted by the mosquito Aedes aegypti. Mosquitoes become infected after ingesting a viremic bloodmeal, and molecular mechanisms involved in bloodmeal digestion may affect the ability of DENV to infect the midgut. We used RNA interference (RNAi) to silence expression of four midgut serine proteases and assessed the effect of each RNAi phenotype on DENV-2 infectivity of Aedes aegypti. Silencing resulted in significant reductions in protease mRNA levels and correlated with a reduction in activity except in the case of late trypsin. RNA silencing of chymotrypsin, early and late trypsin had no effect on DENV-2 infectivity. However, silencing of 5G1 or the addition of soybean trypsin inhibitor to the infectious bloodmeals significantly increased midgut infection rates. These results suggest that some midgut serine proteases may actually limit DENV-2 infectivity of Ae. aegypti.
Author Notes
- INTRODUCTION
- MATERIALS AND METHODS
- Aedes aegypti rearing.
- Dengue virus Type 2 production.
- RNA extraction and cDNA synthesis
- Double stranded RNA (dsRNA) synthesis.
- dsRNA injection and artificial bloodfeeds.
- Quantitative reverse transcriptase polymerase chain reactions (Q-RT-PCR).
- Enzymatic activity assays.
- Trypsin inhibitors.
- Indirect immunofluorescence assays (IFA).
- Statistical analysis.
- RESULTS
- DISCUSSION
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1
Gubler DJ, 1996. The global resurgence of arboviral diseases. Trans R Soc Trop Med Hyg 90 :449–451.
-
2
Monath T, 1994. Dengue: the risk to developed and developing countries. Proc Natl Acad Sci USA 91 :2395–2400.
-
3
Nelson M, 1986. Aedes aegypti: Biology and Ecology. Washington DC: Pan American Health Organization, 1–59.
-
4
Tinker M, 1964. Larval habitat of Aedes aegypti (L.) in the United States. Mosq News 24 :426–432.
-
5
Felix CR, Betschart B, Billingsley PF, Freyvogel TA, 1991. Post-feeding induction of trypsin in the midgut of Aedes aegypti L (Diptera, Culicidae) is separable into 2 cellular phases. Insect Biochem 21 :197–203.
-
6
Noriega FG, Wang XY, Pennington JE, Barillas-Mury CV, Wells MA, 1996. Early trypsin, a female-specific midgut protease in Aedes aegypti: isolation, amino-terminal sequence determination, and cloning and sequencing of the gene. Insect Biochem Mol Biol 26 :119–126.
-
7
Pennington JE, Noriega FG, Wells MA, 1995. The expression of early trypsin in Aedes aegypti. J Cell Biochem 21A (Suppl.):211.
-
8
Graf R, Binz H, Briegel H, 1988. Monoclonal antibodies as probes for Aedes aegypti trypsin. Insect Biochem 18 :463–470.
-
9
Graf R, Raikhel AS, Brown MR, Lea AO, Briegel H, 1986. Mosquito trypsin immunocytochemical localization in the midgut of bloodfed Aedes aegypti (L). Cell Tissue Res 245 :19–27.
-
10
Barillas-Mury C, Graf R, Hagedorn HH, Wells MA, 1991. cDNA and deduced amino acid sequence of a blood meal-induced trypsin from the mosquito, Aedes aegypti. Insect Biochem 21 :825–831.
-
11
Kalhok SE, Tabak LM, Prosser DE, Brook W, Downe AE, White BN, 1993. Isolation, sequencing and characterization of two cDNA clones coding for trypsin-like enzymes from the midgut of Aedes aegypti. Insect Mol Biol 2 :71–79.
-
12
Jiang QJ, Hall M, Noriega FG, Wells M, 1997. cDNA cloning and pattern of expression of an adult, female specific chymotrypsin from Aedes aegypti midgut. Insect Biochem Mol Biol 27 :283–289.
-
13
Black WC, Bennett KE, Gorrochotegui-Escalante N, Barillas-Mury CV, Fernandez-Salas I, Munoz MD, Farfan-Ale JA, Olson KE, Beaty BJ, 2002. Flavivirus susceptibility in Aedes aegypti. Arch Med Res 33 :379–388.
-
14
Noriega FG, Wells MA, 1999. A molecular view of trypsin synthesis in the midgut of Aedes aegypti. J Insect Physiol 45 :613–620.
-
15
Pennington J, Wells M, 2005. The adult midgut: structure and function. Marquardt W, Black WC IV, Freier J, Hagerdorn H, Hemingway J, Higgs S, James A, Kondratieff B, Moore C, eds. Biology of Disease Vectors. Burlington, MA: Elsevier Academic Press, 289–295.
-
16
Espejo RT, Lopez S, Arias C, 1981. Structural polypeptides of simian rotavirus Sa11 and the effect of trypsin. J Virol 37 :156–160.
-
17
Estes MK, Graham DY, Mason BB, 1981. Proteolytic enhancement of rotavirus infectivity—molecular mechanisms. J Virol 39 :879–888.
-
18
Ludwig GV, Christensen BM, Yuill TM, Schultz KT, 1989. Enzyme processing of La Crosse virus glycoprotein G1: a bunyavirus-vector infection model. Virology 171 :108–113.
-
19
Mertens PP, Burroughs JN, Walton A, Wellby MP, Fu H, OHara ‘ RS, Brookes SM, Mellor PS, 1996. Enhanced infectivity of modified bluetongue virus particles for two insect cell lines and for two Culicoides vector species. Virology 217 :582–593.
-
20
Bennett KE, Flick D, Fleming KH, Jochim R, Beaty BJ, Black WC IV, 2005. Quantitative trait loci that control dengue-2 virus dissemination in the mosquito Aedes aegypti. Genetics 170 :185–194.
-
21
Bosio CF, Fulton RE, Salasek ML, Beaty BJ, Black WC IV, 2000. Quantitative trait loci that control vector competence for dengue-2 virus in the mosquito Aedes aegypti. Genetics 156 :687–698.
-
22
Molina-Cruz A, Gupta L, Richardson J, Bennett K, Black WC IV, Barillas-Mury C, 2005. Effect of mosquito midgut trypsin activity on dengue-2 virus and dissemination in Aedes aegypti. Am J Trop Med Hyg 72 :631–637.
-
23
Pierro DJ, Salazar MI, Beaty BJ, Olson KE, 2006. Infectious clone construction of dengue virus type 2, strain Jamaican 1409, and characterization of a conditional E6 mutation. J Gen Virol 87 :2263–2268.
-
24
Keene KM, Foy BD, Sanchez-Vargas I, Beaty BJ, Blair CD, Olson KE, 2004. RNA interference acts as a natural antiviral response to O’nyong-nyong virus (Alphavirus; Togaviridae) infection of Anopheles gambiae. Proc Natl Acad Sci USA 101 :17240–17245.
-
25
Bennett K, Olson K, Munoz Mde L, Fernandez-Salas I, Farfan-Ale J, Higgs S, Black WC IV, Beaty B, 2002. Variation in vector competence for dengue 2 virus among 24 collections of Aedes aegypti from Mexico and the United States. Am J Trop Med Hyg 67 :85–92.
-
26
Lu SJ, Pennington JE, Stonehouse AR, Mobula MM, Wells MA, 2006. Reevaluation of the role of early trypsin activity in the transcriptional activation of the late trypsin gene in the mosquito Aedes aegypti. Insect Biochem Mol Biol 36 :336–343.
-
27
Houk EJ, Kramer LD, Hardy JL, Chiles RE, 1985. Western equine encephalomyelitis virus - In vivo infection and morphogenesis in mosquito mesenteronal epithelial cells. Virus Res 2 :123–138.
-
28
Whitfield SG, Murphy FA, Sudia WD, 1973. St. Louis encephalitis virus: An ultrastructural study of infection in a mosquito vector. Virology 56 :70–87.
-
29
Brown SE, Severson DW, Smith LA, Knudson DL, 2001. Integration of the Aedes aegypti mosquito genetic linkage and physical maps. Genetics 157 :1299–1305.
-
30
Hacker JK, Volkman LE, Hardy JL, 1995. Requirement for the G1-protein of California encephalitis virus in infection in vitro and in vivo. Virology 206 :945–953.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 260 | 215 | 16 |
| Full Text Views | 280 | 9 | 0 |
| PDF Downloads | 109 | 10 | 0 |