Author:
- Introduction
- Materials and Methods
- Plasmid construction.
- Mosquito cell maintenance and transfection.
- Expression and intracellular localization of BmK IT1-GFP fusion proteins.
- Recombinant virus production.
- Mosquito maintenance and transduction.
- Reverse transcription–polymerase chain reaction.
- SDS-PAGE and immunoblots.
- Quantitative real-time PCR.
- Insecticidal efficacy of the recombionant densoviruses against Ae. albopictus.
- Statistical analysis.
- Results
- Discussion
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Attaran A, Roberts DR, Curtis CF, Kilama WL, 2000. Balancing risks on the backs of the poor. Nat Med 6: 729–731.
-
2.
Carlson JO, 1996. Genetic manipulation of mosquitoes: an approach to controlling disease. Trends Biotechnol 14: 447–448.
-
3.
Corsini J, Traul DL, Wilcox CL, Gaines P, Carlson JO, 1996. Efficiency of transduction by recombinant Sindbis replicon virus varies among cell lines, including mosquito cells and rat sensory neurons. Biotechniques 21: 492–497.
-
4.
Ward TW, Kimmick MW, Afanasiev BN, Carlson JO, 2001. Characterization of the structural gene promoter of Aedes aegypti densovirus. J Virol 75: 1325–1331.
-
5.
Lebedeva PO, Zelenko AP, Kuznetsova MA, Gudzgorban AP, 1972. Studies on the demonstration of a viral infection in larvae of Aedes aegypti mosquitoes. Microbiol JSU 34: 70–73.
-
6.
Carlson J, Suchman E, Buchatsky L, 2006. Densoviruses for control and genetic manipulation of mosquitoes. Adv Virus Res 68: 361–392.
-
7.
Pattanakitsakul SN, Boonnak K, Auethavornanan K, Jairungsri A, Duangjinda T, Puttatesk P, Thongrungkiat S, Malasit P, 2007. A new densovirus isolated from the mosquito Toxorhynchites splendens (Wiedemann) (Diptera: Culicidae). Southeast Asian J Trop Med Public Health 38: 283–293.
-
8.
Ren X, Hoiczyk E, Rasgon JL, 2008. Viral paratransgenesis in the malaria vector Anopheles gambiae. PLoS Pathog 4: e1000135.
-
9.
Zhai YG, Lv XJ, Sun XH, Fu SH, Gong ZD, Fen Y, Tong SX, Wang ZX, Tang Q, Attoui H, Liang GD, 2008. Isolation and characterization of the full coding sequence of a novel densovirus from the mosquito Culex pipiens pallens. J Gen Virol 89: 195–199.
-
10.
Sivaram A, Barde PV, Kumar SR, Yadav P, Gokhale MD, Basu A, Mourya DT, 2009. Isolation and characterization of densonucleosis virus from Aedes aegypti mosquitoes and its distribution in India. Intervirology 52: 1–7.
-
11.
van Beek NA, Hughes PR, 1998. The response time of insect larvae infected with recombinant baculoviruses. J Invertebr Pathol 72: 338–347.
-
12.
McCutchen BF, Choudary PV, Crenshaw R, Maddox D, Kamita SG, Palekar N, Volrath S, Fowler E, Hammock BD, Maeda S, 1991. Development of a recombinant baculovirus expressing an insect-selective neurotoxin: potential for pest control. Biotechnology (NY) 9: 848–852.
-
13.
Jiang H, Zhang JM, Wang JP, Yang B, Liu CF, Lu J, Hu YY, 2007. Genetic engineering of Periplaneta fuliginosa densovirus as an improved biopesticide. Arch Virol 152: 383–394.
-
14.
Goudet C, Chi CW, Tytgat J, 2002. An overview of toxins and genes from the venom of the Asian scorpion Buthus martensi Karsch. Toxicon 40: 1239–1258.
-
15.
Ward TW, Jenkins MS, Afanasiev BN, Edwards M, Duda BA, Suchman E, Jacobs-Lorena M, Beaty BJ, Carlson JO, 2001. Aedes aegypti transducing densovirus pathogenesis and expression in Aedes aegypti and Anopheles gambiae larvae. Insect Mol Biol 10: 397–405.
-
16.
Lu P, Feng MG, 2008. Bifunctional enhancement of a beta-glucanase-xylanase fusion enzyme by optimization of peptide linkers. Appl Microbiol Biotechnol 79: 579–587.
-
17.
Afanasiev BN, Kozlov YV, Carlson JO, Beaty BJ, 1994. Densovirus of Aedes aegypti as an expression vector in mosquito cells. Exp Parasitol 79: 322–339.
-
18.
Afanasiev BN, Ward TW, Beaty BJ, Carlson JO, 1999. Transduction of Aedes aegypti mosquitoes with vectors derived from Aedes densovirus. Virology 257: 62–72.
-
19.
Walker SL, Wonderling RS, Owens RA, 1997. Mutational analysis of the adeno-associated virus Rep68 protein: identification of critical residues necessary for site-specific endonuclease activity. J Virol 71: 2722–2730.
-
20.
Wilson GM, Jindal HK, Yeung DE, Chen W, Astell CR, 1991. Expression of minute virus of mice major nonstructural protein in insect cells: purification and identification of ATPase and helicase activities. Virology 185: 90–98.
-
21.
Yang B, Zhang J, Cai D, Li D, Chen W, Jiang H, Hu Y, 2006. Biochemical characterization of Periplaneta fuliginosa densovirus non-structural protein NS1. Biochem Biophys Res Commun 342: 1188–1196.
-
22.
You H, Liu Y, Prasad CK, Agrawal N, Zhang D, Bandyopadhyay S, Liu H, Kay HH, Mehta JL, Hermonat PL, 2006. Multiple human papillomavirus genes affect the adeno-associated virus life cycle. Virology 344: 532–540.
-
23.
Zlotkin E, Miranda F, Kupeyan C, Lissitzky S, 1971. A new toxic protein in the venom of the scorpion Androctonus australis Hector. Toxicon 9: 9–13.
-
24.
Darbon H, Zlotkin E, Kopeyan C, van Rietschoten J, Rochat H, 1982. Covalent structure of the insect toxin of the North African scorpion Androctonus australis Hector. Int J Pept Protein Res 20: 320–330.
-
25.
Allen-Miura TM, Afanasiev BN, Olson KE, Beaty BJ, Carlson JO, 1999. Packaging of AeDNV-GFP transducing virus by expression of densovirus structural proteins from a sindbis virus expression system. Virology 257: 54–61.
-
26.
Kimmick MW, Afanasiev BN, Beaty BJ, Carlson JO, 1998. Gene expression and regulation from the p7 promoter of Aedes densonucleosis virus. J Virol 72: 4364–4370.
-
27.
Dingwall C, Robbins J, Dilworth SM, Roberts B, Richardson WD, 1988. The nucleoplasmin nuclear location sequence is larger and more complex than that of SV-40 large T antigen. J Cell Biol 107: 841–849.
-
28.
Christensen J, Tattersall P, 2002. Parvovirus initiator protein NS1 and RPA coordinate replication fork progression in a reconstituted DNA replication system. J Virol 76: 6518–6531.
-
29.
Jindal HK, Yong CB, Wilson GM, Tam P, Astell CR, 1994. Mutations in the NTP-binding motif of minute virus of mice (MVM) NS-1 protein uncouple ATPase and DNA helicase functions. J Biol Chem 269: 3283–3289.
-
30.
Vendeville A, Ravallec M, Jousset FX, Devise M, Mutuel D, Lopez-Ferber M, Fournier P, Dupressoir T, Ogliastro M, 2009. Densovirus infectious pathway requires clathrin-mediated endocytosis followed by trafficking to the nucleus. J Virol 83: 4678–4689.
-
31.
Zlotkin E, Fishman Y, Elazar M, 2000. AaIT: from neurotoxin to insecticide. Biochimie 82: 869–881.
-
32.
Higgs S, Olson KE, Klimowski L, Powers AM, Carlson JO, Possee RD, Beaty BJ, 1995. Mosquito sensitivity to a scorpion neurotoxin expressed using an infectious Sindbis virus vector. Insect Mol Biol 4: 97–103.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 1760 | 1626 | 51 |
| Full Text Views | 457 | 13 | 0 |
| PDF Downloads | 206 | 13 | 0 |
A Recombinant AeDNA Containing the Insect-Specific Toxin, BmK IT1, Displayed an Increasing Pathogenicity on Aedes albopictus
Jin-Bao Gu
Jin-Bao Gu
School of Public Health and Tropical Medicine, Southern Medical University, Guangdong, People's Republic of China; Institute of Genetic Engineering, Southern Medical University, Guangdong, People's Republic of China; School of Public Health and Tropical Medicine, Southern Medical University, Guangdong, People's Republic of China
Search for other papers by Jin-Bao Gu in
Current site
Google Scholar
PubMed
Search for other papers by Jin-Bao Gu in
Current site
Google Scholar
PubMed
Yun-Qiao Dong
Yun-Qiao Dong
School of Public Health and Tropical Medicine, Southern Medical University, Guangdong, People's Republic of China; Institute of Genetic Engineering, Southern Medical University, Guangdong, People's Republic of China; School of Public Health and Tropical Medicine, Southern Medical University, Guangdong, People's Republic of China
Search for other papers by Yun-Qiao Dong in
Current site
Google Scholar
PubMed
Search for other papers by Yun-Qiao Dong in
Current site
Google Scholar
PubMed
Hong-Juan Peng
Hong-Juan Peng
School of Public Health and Tropical Medicine, Southern Medical University, Guangdong, People's Republic of China; Institute of Genetic Engineering, Southern Medical University, Guangdong, People's Republic of China; School of Public Health and Tropical Medicine, Southern Medical University, Guangdong, People's Republic of China
Search for other papers by Hong-Juan Peng in
Current site
Google Scholar
PubMed
Search for other papers by Hong-Juan Peng in
Current site
Google Scholar
PubMed
Xiao-Guang Chen
Xiao-Guang Chen
School of Public Health and Tropical Medicine, Southern Medical University, Guangdong, People's Republic of China; Institute of Genetic Engineering, Southern Medical University, Guangdong, People's Republic of China; School of Public Health and Tropical Medicine, Southern Medical University, Guangdong, People's Republic of China
Search for other papers by Xiao-Guang Chen in
Current site
Google Scholar
PubMed
Search for other papers by Xiao-Guang Chen in
Current site
Google Scholar
PubMed
The Aedes aegypti densovirus (AeDNV) has previously shown potential in mosquito control. To improve its efficacy as a biopesticide, the gene for an excitatory insect-specific toxin from Buthus martensii Karsch (BmK IT1) was inserted into the AeDNV genome and cloned into pUCA plasmid. The coding sequence for green fluorescent protein was ligated to the C-terminus of the BmK IT1 gene as a screening marker. Recombinant and helper plasmids were cotransfected into C6/36 cells; wild-type viruses were the controls. The recombinant viruses were identified and quantified by real-time polymerase chain reaction and exposed to Ae. albopictus larvae for the evaluation of its bioinsecticidal activity. LT50 and LD50 bioassays showed that the recombinant AeDNV had stronger and faster pathogenic effects on Ae. albopictus than the wild-type virus. This is the first report on the recombinant AeDNA containing the insect-specific toxin, BmK IT1, which may be used to develop a novel type of insecticide.
Author Notes
Financial support: The research was supported by the National Natural Science Foundation of China (no. 30800957 and 30771871) and President Foundation of School of Public Health and Tropical Medicine, Southern Medical University (no. GW200829).
Authors' addresses: Jin-Bao Gu, Hong-Juan Peng, and Xiao-Guang Chen, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, China, E-mails: gujinbao@fimmu.com, floriapeng@hotmail.com, and xgchen@fimmu.com. Yun-Qiao Dong, Institute of Genetic Engineering, Southern Medical University, Guangzhou, China, E-mail: yunqiao@fimmu.com.
- Introduction
- Materials and Methods
- Plasmid construction.
- Mosquito cell maintenance and transfection.
- Expression and intracellular localization of BmK IT1-GFP fusion proteins.
- Recombinant virus production.
- Mosquito maintenance and transduction.
- Reverse transcription–polymerase chain reaction.
- SDS-PAGE and immunoblots.
- Quantitative real-time PCR.
- Insecticidal efficacy of the recombionant densoviruses against Ae. albopictus.
- Statistical analysis.
- Results
- Discussion
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
Attaran A, Roberts DR, Curtis CF, Kilama WL, 2000. Balancing risks on the backs of the poor. Nat Med 6: 729–731.
-
2.
Carlson JO, 1996. Genetic manipulation of mosquitoes: an approach to controlling disease. Trends Biotechnol 14: 447–448.
-
3.
Corsini J, Traul DL, Wilcox CL, Gaines P, Carlson JO, 1996. Efficiency of transduction by recombinant Sindbis replicon virus varies among cell lines, including mosquito cells and rat sensory neurons. Biotechniques 21: 492–497.
-
4.
Ward TW, Kimmick MW, Afanasiev BN, Carlson JO, 2001. Characterization of the structural gene promoter of Aedes aegypti densovirus. J Virol 75: 1325–1331.
-
5.
Lebedeva PO, Zelenko AP, Kuznetsova MA, Gudzgorban AP, 1972. Studies on the demonstration of a viral infection in larvae of Aedes aegypti mosquitoes. Microbiol JSU 34: 70–73.
-
6.
Carlson J, Suchman E, Buchatsky L, 2006. Densoviruses for control and genetic manipulation of mosquitoes. Adv Virus Res 68: 361–392.
-
7.
Pattanakitsakul SN, Boonnak K, Auethavornanan K, Jairungsri A, Duangjinda T, Puttatesk P, Thongrungkiat S, Malasit P, 2007. A new densovirus isolated from the mosquito Toxorhynchites splendens (Wiedemann) (Diptera: Culicidae). Southeast Asian J Trop Med Public Health 38: 283–293.
-
8.
Ren X, Hoiczyk E, Rasgon JL, 2008. Viral paratransgenesis in the malaria vector Anopheles gambiae. PLoS Pathog 4: e1000135.
-
9.
Zhai YG, Lv XJ, Sun XH, Fu SH, Gong ZD, Fen Y, Tong SX, Wang ZX, Tang Q, Attoui H, Liang GD, 2008. Isolation and characterization of the full coding sequence of a novel densovirus from the mosquito Culex pipiens pallens. J Gen Virol 89: 195–199.
-
10.
Sivaram A, Barde PV, Kumar SR, Yadav P, Gokhale MD, Basu A, Mourya DT, 2009. Isolation and characterization of densonucleosis virus from Aedes aegypti mosquitoes and its distribution in India. Intervirology 52: 1–7.
-
11.
van Beek NA, Hughes PR, 1998. The response time of insect larvae infected with recombinant baculoviruses. J Invertebr Pathol 72: 338–347.
-
12.
McCutchen BF, Choudary PV, Crenshaw R, Maddox D, Kamita SG, Palekar N, Volrath S, Fowler E, Hammock BD, Maeda S, 1991. Development of a recombinant baculovirus expressing an insect-selective neurotoxin: potential for pest control. Biotechnology (NY) 9: 848–852.
-
13.
Jiang H, Zhang JM, Wang JP, Yang B, Liu CF, Lu J, Hu YY, 2007. Genetic engineering of Periplaneta fuliginosa densovirus as an improved biopesticide. Arch Virol 152: 383–394.
-
14.
Goudet C, Chi CW, Tytgat J, 2002. An overview of toxins and genes from the venom of the Asian scorpion Buthus martensi Karsch. Toxicon 40: 1239–1258.
-
15.
Ward TW, Jenkins MS, Afanasiev BN, Edwards M, Duda BA, Suchman E, Jacobs-Lorena M, Beaty BJ, Carlson JO, 2001. Aedes aegypti transducing densovirus pathogenesis and expression in Aedes aegypti and Anopheles gambiae larvae. Insect Mol Biol 10: 397–405.
-
16.
Lu P, Feng MG, 2008. Bifunctional enhancement of a beta-glucanase-xylanase fusion enzyme by optimization of peptide linkers. Appl Microbiol Biotechnol 79: 579–587.
-
17.
Afanasiev BN, Kozlov YV, Carlson JO, Beaty BJ, 1994. Densovirus of Aedes aegypti as an expression vector in mosquito cells. Exp Parasitol 79: 322–339.
-
18.
Afanasiev BN, Ward TW, Beaty BJ, Carlson JO, 1999. Transduction of Aedes aegypti mosquitoes with vectors derived from Aedes densovirus. Virology 257: 62–72.
-
19.
Walker SL, Wonderling RS, Owens RA, 1997. Mutational analysis of the adeno-associated virus Rep68 protein: identification of critical residues necessary for site-specific endonuclease activity. J Virol 71: 2722–2730.
-
20.
Wilson GM, Jindal HK, Yeung DE, Chen W, Astell CR, 1991. Expression of minute virus of mice major nonstructural protein in insect cells: purification and identification of ATPase and helicase activities. Virology 185: 90–98.
-
21.
Yang B, Zhang J, Cai D, Li D, Chen W, Jiang H, Hu Y, 2006. Biochemical characterization of Periplaneta fuliginosa densovirus non-structural protein NS1. Biochem Biophys Res Commun 342: 1188–1196.
-
22.
You H, Liu Y, Prasad CK, Agrawal N, Zhang D, Bandyopadhyay S, Liu H, Kay HH, Mehta JL, Hermonat PL, 2006. Multiple human papillomavirus genes affect the adeno-associated virus life cycle. Virology 344: 532–540.
-
23.
Zlotkin E, Miranda F, Kupeyan C, Lissitzky S, 1971. A new toxic protein in the venom of the scorpion Androctonus australis Hector. Toxicon 9: 9–13.
-
24.
Darbon H, Zlotkin E, Kopeyan C, van Rietschoten J, Rochat H, 1982. Covalent structure of the insect toxin of the North African scorpion Androctonus australis Hector. Int J Pept Protein Res 20: 320–330.
-
25.
Allen-Miura TM, Afanasiev BN, Olson KE, Beaty BJ, Carlson JO, 1999. Packaging of AeDNV-GFP transducing virus by expression of densovirus structural proteins from a sindbis virus expression system. Virology 257: 54–61.
-
26.
Kimmick MW, Afanasiev BN, Beaty BJ, Carlson JO, 1998. Gene expression and regulation from the p7 promoter of Aedes densonucleosis virus. J Virol 72: 4364–4370.
-
27.
Dingwall C, Robbins J, Dilworth SM, Roberts B, Richardson WD, 1988. The nucleoplasmin nuclear location sequence is larger and more complex than that of SV-40 large T antigen. J Cell Biol 107: 841–849.
-
28.
Christensen J, Tattersall P, 2002. Parvovirus initiator protein NS1 and RPA coordinate replication fork progression in a reconstituted DNA replication system. J Virol 76: 6518–6531.
-
29.
Jindal HK, Yong CB, Wilson GM, Tam P, Astell CR, 1994. Mutations in the NTP-binding motif of minute virus of mice (MVM) NS-1 protein uncouple ATPase and DNA helicase functions. J Biol Chem 269: 3283–3289.
-
30.
Vendeville A, Ravallec M, Jousset FX, Devise M, Mutuel D, Lopez-Ferber M, Fournier P, Dupressoir T, Ogliastro M, 2009. Densovirus infectious pathway requires clathrin-mediated endocytosis followed by trafficking to the nucleus. J Virol 83: 4678–4689.
-
31.
Zlotkin E, Fishman Y, Elazar M, 2000. AaIT: from neurotoxin to insecticide. Biochimie 82: 869–881.
-
32.
Higgs S, Olson KE, Klimowski L, Powers AM, Carlson JO, Possee RD, Beaty BJ, 1995. Mosquito sensitivity to a scorpion neurotoxin expressed using an infectious Sindbis virus vector. Insect Mol Biol 4: 97–103.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 1760 | 1626 | 51 |
| Full Text Views | 457 | 13 | 0 |
| PDF Downloads | 206 | 13 | 0 |