Martina BE, Koraka P, Osterhaus AD, 2009. Dengue virus pathogenesis: an integrated view. Clin Microbiol Rev 22: 564–581.
Weaver SC, Reisen WK, 2010. Present and future arboviral threats. Antiviral Res 85: 328–345.
Gubler DJ, 2006. Dengue/dengue haemorrhagic fever: history and current status. Novartis Found Symp 277: 3–16.
Jarman RG, Holmes EC, Rodpradit P, Klungthong C, Gibbons RV, Nisalak A, Rothman AL, Libraty DH, Ennis FA, Mammen MP Jr, Endy TP, 2008. Microevolution of dengue viruses circulating among primary school children in Kamphaeng Phet, Thailand. J Virol 82: 5494–5500.
Weaver SC, Vasilakis N, 2009. Molecular evolution of dengue viruses: contributions of phylogenetics to understanding the history and epidemiology of the preeminent arboviral disease. Infect Genet Evol 9: 523–540.
Alto BW, Reiskind MH, Lounibos LP, 2008. Size alters susceptibility of vectors to dengue virus infection and dissemination. Am J Trop Med Hyg 79: 688–695.
Alto BW, Lounibos LP, Mores CN, Reiskind MH, 2008. Larval competition alters susceptibility of adult Aedes mosquitoes to dengue infection. Proc Biol Sci 275: 463–471.
Knox TB, Kay BH, Hall RA, Ryan PA, 2003. Enhanced vector competence of Aedes aegypti (Diptera: Culicidae) from the Torres Strait compared with mainland Australia for dengue 2 and 4 viruses. J Med Entomol 40: 950–956.
Bennett KE, Olson KE, Munoz M de L, Fernandez-Salas I, Farfan-Ale JA, Higgs S, Black WC 4th, Beaty BJ, 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.
Black WC 4th, Bennett KE, Gorrochotegui-Escalante N, Barillas-Mury CV, Fernandez-Salas I, de Lourdes Munoz M, Farfan-Ale JA, Olson KE, Beaty BJ, 2002. Flavivirus susceptibility in Aedes aegypti. Arch Med Res 33: 379–388.
Xi Z, Ramirez JL, Dimopoulos G, 2008. The Aedes aegypti toll pathway controls dengue virus infection. PLoS Pathog 4: e1000098.
Fragkoudis R, Attarzadeh-Yazdi G, Nash AA, Fazakerley JK, Kohl A, 2009. Advances in dissecting mosquito innate immune responses to arbovirus infection. J Gen Virol 90: 2061–2072.
Sanchez-Vargas I, Travanty EA, Keene KM, Franz AW, Beaty BJ, Blair CD, Olson KE, 2004. RNA interference, arthropod-borne viruses, and mosquitoes. Virus Res 102: 65–74.
Sanchez-Vargas I, Scott JC, Poole-Smith BK, Franz AW, Barbosa-Solomieu V, Wilusz J, Olson KE, Blair CD, 2009. Dengue virus type 2 infections of Aedes aegypti are modulated by the mosquito's RNA interference pathway. PLoS Pathog 5: e1000299.
Champagne DE, Smartt CT, Ribeiro JM, James AA, 1995. The salivary gland-specific apyrase of the mosquito Aedes aegypti is a member of the 5′-nucleotidase family. Proc Natl Acad Sci USA 92: 694–698.
Valenzuela JG, Pham VM, Garfield MK, Francischetti IM, Ribeiro JM, 2002. Toward a description of the sialome of the adult female mosquito Aedes aegypti. Insect Biochem Mol Biol 32: 1101–1122.
Ribeiro JM, Arca B, Lombardo F, Calvo E, Phan VM, Chandra PK, Wikel SK, 2007. An annotated catalogue of salivary gland transcripts in the adult female mosquito, Aedes aegypti. BMC Genomics 8: 6.
Smartt CT, Kim AP, Grossman GL, James AA, 1995. The Apyrase gene of the vector mosquito, Aedes aegypti, is expressed specifically in the adult female salivary glands. Exp Parasitol 81: 239–248.
Sim S, Ramirez JL, Dimopoulos G, 2012. Dengue virus infection of the Aedes aegypti salivary gland and chemosensory apparatus induces genes that modulate infection and blood-feeding behavior. PLoS Pathog 8: e1002631.
Almeras L, Fontaine A, Belghazi M, Bourdon S, Boucomont-Chapeaublanc E, Orlandi-Pradines E, Baragatti M, Corre-Catelin N, Reiter P, Pradines B, Fusai T, Rogier C, 2010. Salivary gland protein repertoire from Aedes aegypti mosquitoes. Vector Borne Zoonotic Dis 10: 391–402.
Almeras L, Orlandi-Pradines E, Fontaine A, Villard C, Boucomont E, de Senneville LD, Baragatti M, Pascual A, Pradines B, Corre-Catelin N, Pages F, Reiter P, Rogier C, Fusai T, 2009. Sialome individuality between Aedes aegypti colonies. Vector Borne Zoonotic Dis 9: 531–541.
Ribeiro JM, Francischetti IM, 2003. Role of arthropod saliva in blood feeding: sialome and post-sialome perspectives. Annu Rev Entomol 48: 73–88.
Ribeiro JM, 1995. Blood-feeding arthropods: live syringes or invertebrate pharmacologists? Infect Agents Dis 4: 143–152.
Guo X, Xu Y, Bian G, Pike AD, Xie Y, Xi Z, 2010. Response of the mosquito protein interaction network to dengue infection. BMC Genomics 11: 380.
Wasinpiyamongkol L, Patramool S, Thongrungkiat S, Maneekan P, Sangmukdanan S, Misse D, Luplertlop N, 2012. Protein expression in the salivary glands of dengue-infected Aedes aegypti mosquitoes and blood-feeding success. Southeast Asian J Trop Med Public Health 43: 1346–1357.
Tchankouo-Nguetcheu S, Bourguet E, Lenormand P, Rousselle JC, Namane A, Choumet V, 2012. Infection by chikungunya virus modulates the expression of several proteins in Aedes aegypti salivary glands. Parasit Vectors 5: 264.
Chisenhall DM, Mores CN, 2009. Diversification of West Nile virus in a subtropical region. Virol J 6: 106.
Megy K, Emrich SJ, Lawson D, Campbell D, Dialynas E, Hughes DS, Koscielny G, Louis C, Maccallum RM, Redmond SN, Sheehan A, Topalis P, Wilson D, 2012. VectorBase: improvements to a bioinformatics resource for invertebrate vector genomics. Nucleic Acids Res 40: D729–D734.
Bonizzoni M, Dunn WA, Campbell CL, Olson KE, Marinotti O, James AA, 2012. Complex modulation of the Aedes aegypti transcriptome in response to dengue virus infection. PLoS One 7: e50512.
Sim S, Dimopoulos G, 2010. Dengue virus inhibits immune responses in Aedes aegypti cells. PLoS One 5: e10678.
Dostert C, Jouanguy E, Irving P, Troxler L, Galiana-Arnoux D, Hetru C, Hoffmann JA, Imler JL, 2005. The Jak-STAT signaling pathway is required but not sufficient for the antiviral response of drosophila. Nat Immunol 6: 946–953.
Sabatier L, Jouanguy E, Dostert C, Zachary D, Dimarcq JL, Bulet P, Imler JL, 2003. Pherokine-2 and -3. Eur J Biochem 270: 3398–3407.
Mounsey A, Bauer P, Hope IA, 2002. Evidence suggesting that a fifth of annotated Caenorhabditis elegans genes may be pseudogenes. Genome Res 12: 770–775.
Platt KB, Linthicum KJ, Myint KS, Innis BL, Lerdthusnee K, Vaughn DW, 1997. Impact of dengue virus infection on feeding behavior of Aedes aegypti. Am J Trop Med Hyg 57: 119–125.
Salazar MI, Richardson JH, Sanchez-Vargas I, Olson KE, Beaty BJ, 2007. Dengue virus type 2: replication and tropisms in orally infected Aedes aegypti mosquitoes. BMC Microbiol 7: 9.
Lawson D, Arensburger P, Atkinson P, Besansky NJ, Bruggner RV, Butler R, Campbell KS, Christophides GK, Christley S, Dialynas E, Hammond M, Hill CA, Konopinski N, Lobo NF, MacCallum RM, Madey G, Megy K, Meyer J, Redmond S, Severson DW, Stinson EO, Topalis P, Birney E, Gelbart WM, Kafatos FC, Louis C, Collins FH, 2009. VectorBase: a data resource for invertebrate vector genomics. Nucleic Acids Res 37: D583–D587.
Calvo E, Tokumasu F, Marinotti O, Villeval JL, Ribeiro JM, Francischetti IM, 2007. Aegyptin, a novel mosquito salivary gland protein, specifically binds to collagen and prevents its interaction with platelet glycoprotein VI, integrin alpha2beta1, and von Willebrand factor. J Biol Chem 282: 26928–26938.
Rossingol PA, Spielman A, 1982. Fluid transport across the ducts of a mosquito. J Insect Physiol 28: 579–583.
Peng Z, Simons FE, 2007. Advances in mosquito allergy. Curr Opin Allergy Clin Immunol 7: 350–354.
Simons FE, Peng Z, 2001. Mosquito allergy: recombinant mosquito salivary antigens for new diagnostic tests. Int Arch Allergy Immunol 124: 403–405.
Sylvestre G, Gandini M, Maciel-de-Freitas R, 2013. Age-dependent effects of oral infection with dengue virus on Aedes aegypti (Diptera: Culicidae) feeding behavior, survival, oviposition success and fecundity. PLoS One 8: e59933.
Ramirez JL, Dimopoulos G, 2010. The Toll immune signaling pathway control conserved anti-dengue defenses across diverse Ae. aegypti strains and against multiple dengue virus serotypes. Dev Comp Immunol 34: 625–629.
Linthicum KJ, Platt K, Myint KS, Lerdthusnee K, Innis BL, Vaughn DW, 1996. Dengue 3 virus distribution in the mosquito Aedes aegypti: an immunocytochemical study. Med Vet Entomol 10: 87–92.
Mackenzie J, 2005. Wrapping things up about virus RNA replication. Traffic 6: 967–977.
Fernandez-Garcia MD, Mazzon M, Jacobs M, Amara A, 2009. Pathogenesis of flavivirus infections: using and abusing the host cell. Cell Host Microbe 5: 318–328.
Jin JP, Chong SM, 2010. Localization of the two tropomyosin-binding sites of troponin T. Arch Biochem Biophys 500: 144–150.
Ng ML, Hong SS, 1989. Flavivirus infection: essential ultrastructural changes and association of Kunjin virus NS3 protein with microtubules. Arch Virol 106: 103–120.
Lyles DS, 2000. Cytopathogenesis and inhibition of host gene expression by RNA viruses. Microbiol Mol Biol Rev 64: 709–724.
Hayes JD, Flanagan JU, Jowsey IR, 2005. Glutathione transferases. Annu Rev Pharmacol Toxicol 45: 51–88.
Sheehan D, Meade G, Foley VM, Dowd CA, 2001. Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochem J 360: 1–16.
Gui Z, Hou C, Liu T, Qin G, Li M, Jin B, 2009. Effects of insect viruses and pesticides on glutathione S-transferase activity and gene expression in Bombyx mori. J Econ Entomol 102: 1591–1598.
Drakesmith H, Prentice A, 2008. Viral infection and iron metabolism. Nat Rev Microbiol 6: 541–552.
Past two years | Past Year | Past 30 Days | |
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Abstract Views | 760 | 490 | 21 |
Full Text Views | 440 | 13 | 0 |
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Dengue virus (DENV) is the most important mosquito-transmitted flavivirus that is transmitted throughout the tropical and subtropical regions of the world. The primary mosquito vector of DENV in urban locations is Aedes aegypti. Key to understanding the transmission of DENV is the relationship between pathogen and vector. Accordingly, we report our preliminary characterization of the differentially expressed proteins from Ae. aegypti mosquitoes after DENV infection. We investigated the virus–vector interaction through changes in the proteome of the salivary glands of mosquitoes with disseminated DENV serotype 2 (DENV-2) infections using two-dimensional gel electrophoresis and identification by mass spectrometry. Our findings indicate that DENV-2 infection in the Ae. aegypti salivary gland alters the expression of structural, secreted, and metabolic proteins. These changes in the salivary gland proteome highlight the virally influenced environment caused by a DENV-2 infection and warrant additional investigation to determine if these differences extend to the expectorated saliva.
Financial support: This work was supported by National Institutes of Health Grant P20GM103458.
Authors' addresses: Daniel M. Chisenhall, Berlin L. Londono, Rebecca C. Christofferson, Michael K. McCracken, and Christopher N. Mores, School of Veterinary Medicine, Department of Pathobiological Sciences, Louisiana State University, Baton Rouge, LA, E-mails: dchisenh@lsu.edu, berlin@lsu.edu, rcarri1@lsu.edu, mmccra4@lsu.edu, and cmores@lsu.edu.
Martina BE, Koraka P, Osterhaus AD, 2009. Dengue virus pathogenesis: an integrated view. Clin Microbiol Rev 22: 564–581.
Weaver SC, Reisen WK, 2010. Present and future arboviral threats. Antiviral Res 85: 328–345.
Gubler DJ, 2006. Dengue/dengue haemorrhagic fever: history and current status. Novartis Found Symp 277: 3–16.
Jarman RG, Holmes EC, Rodpradit P, Klungthong C, Gibbons RV, Nisalak A, Rothman AL, Libraty DH, Ennis FA, Mammen MP Jr, Endy TP, 2008. Microevolution of dengue viruses circulating among primary school children in Kamphaeng Phet, Thailand. J Virol 82: 5494–5500.
Weaver SC, Vasilakis N, 2009. Molecular evolution of dengue viruses: contributions of phylogenetics to understanding the history and epidemiology of the preeminent arboviral disease. Infect Genet Evol 9: 523–540.
Alto BW, Reiskind MH, Lounibos LP, 2008. Size alters susceptibility of vectors to dengue virus infection and dissemination. Am J Trop Med Hyg 79: 688–695.
Alto BW, Lounibos LP, Mores CN, Reiskind MH, 2008. Larval competition alters susceptibility of adult Aedes mosquitoes to dengue infection. Proc Biol Sci 275: 463–471.
Knox TB, Kay BH, Hall RA, Ryan PA, 2003. Enhanced vector competence of Aedes aegypti (Diptera: Culicidae) from the Torres Strait compared with mainland Australia for dengue 2 and 4 viruses. J Med Entomol 40: 950–956.
Bennett KE, Olson KE, Munoz M de L, Fernandez-Salas I, Farfan-Ale JA, Higgs S, Black WC 4th, Beaty BJ, 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.
Black WC 4th, Bennett KE, Gorrochotegui-Escalante N, Barillas-Mury CV, Fernandez-Salas I, de Lourdes Munoz M, Farfan-Ale JA, Olson KE, Beaty BJ, 2002. Flavivirus susceptibility in Aedes aegypti. Arch Med Res 33: 379–388.
Xi Z, Ramirez JL, Dimopoulos G, 2008. The Aedes aegypti toll pathway controls dengue virus infection. PLoS Pathog 4: e1000098.
Fragkoudis R, Attarzadeh-Yazdi G, Nash AA, Fazakerley JK, Kohl A, 2009. Advances in dissecting mosquito innate immune responses to arbovirus infection. J Gen Virol 90: 2061–2072.
Sanchez-Vargas I, Travanty EA, Keene KM, Franz AW, Beaty BJ, Blair CD, Olson KE, 2004. RNA interference, arthropod-borne viruses, and mosquitoes. Virus Res 102: 65–74.
Sanchez-Vargas I, Scott JC, Poole-Smith BK, Franz AW, Barbosa-Solomieu V, Wilusz J, Olson KE, Blair CD, 2009. Dengue virus type 2 infections of Aedes aegypti are modulated by the mosquito's RNA interference pathway. PLoS Pathog 5: e1000299.
Champagne DE, Smartt CT, Ribeiro JM, James AA, 1995. The salivary gland-specific apyrase of the mosquito Aedes aegypti is a member of the 5′-nucleotidase family. Proc Natl Acad Sci USA 92: 694–698.
Valenzuela JG, Pham VM, Garfield MK, Francischetti IM, Ribeiro JM, 2002. Toward a description of the sialome of the adult female mosquito Aedes aegypti. Insect Biochem Mol Biol 32: 1101–1122.
Ribeiro JM, Arca B, Lombardo F, Calvo E, Phan VM, Chandra PK, Wikel SK, 2007. An annotated catalogue of salivary gland transcripts in the adult female mosquito, Aedes aegypti. BMC Genomics 8: 6.
Smartt CT, Kim AP, Grossman GL, James AA, 1995. The Apyrase gene of the vector mosquito, Aedes aegypti, is expressed specifically in the adult female salivary glands. Exp Parasitol 81: 239–248.
Sim S, Ramirez JL, Dimopoulos G, 2012. Dengue virus infection of the Aedes aegypti salivary gland and chemosensory apparatus induces genes that modulate infection and blood-feeding behavior. PLoS Pathog 8: e1002631.
Almeras L, Fontaine A, Belghazi M, Bourdon S, Boucomont-Chapeaublanc E, Orlandi-Pradines E, Baragatti M, Corre-Catelin N, Reiter P, Pradines B, Fusai T, Rogier C, 2010. Salivary gland protein repertoire from Aedes aegypti mosquitoes. Vector Borne Zoonotic Dis 10: 391–402.
Almeras L, Orlandi-Pradines E, Fontaine A, Villard C, Boucomont E, de Senneville LD, Baragatti M, Pascual A, Pradines B, Corre-Catelin N, Pages F, Reiter P, Rogier C, Fusai T, 2009. Sialome individuality between Aedes aegypti colonies. Vector Borne Zoonotic Dis 9: 531–541.
Ribeiro JM, Francischetti IM, 2003. Role of arthropod saliva in blood feeding: sialome and post-sialome perspectives. Annu Rev Entomol 48: 73–88.
Ribeiro JM, 1995. Blood-feeding arthropods: live syringes or invertebrate pharmacologists? Infect Agents Dis 4: 143–152.
Guo X, Xu Y, Bian G, Pike AD, Xie Y, Xi Z, 2010. Response of the mosquito protein interaction network to dengue infection. BMC Genomics 11: 380.
Wasinpiyamongkol L, Patramool S, Thongrungkiat S, Maneekan P, Sangmukdanan S, Misse D, Luplertlop N, 2012. Protein expression in the salivary glands of dengue-infected Aedes aegypti mosquitoes and blood-feeding success. Southeast Asian J Trop Med Public Health 43: 1346–1357.
Tchankouo-Nguetcheu S, Bourguet E, Lenormand P, Rousselle JC, Namane A, Choumet V, 2012. Infection by chikungunya virus modulates the expression of several proteins in Aedes aegypti salivary glands. Parasit Vectors 5: 264.
Chisenhall DM, Mores CN, 2009. Diversification of West Nile virus in a subtropical region. Virol J 6: 106.
Megy K, Emrich SJ, Lawson D, Campbell D, Dialynas E, Hughes DS, Koscielny G, Louis C, Maccallum RM, Redmond SN, Sheehan A, Topalis P, Wilson D, 2012. VectorBase: improvements to a bioinformatics resource for invertebrate vector genomics. Nucleic Acids Res 40: D729–D734.
Bonizzoni M, Dunn WA, Campbell CL, Olson KE, Marinotti O, James AA, 2012. Complex modulation of the Aedes aegypti transcriptome in response to dengue virus infection. PLoS One 7: e50512.
Sim S, Dimopoulos G, 2010. Dengue virus inhibits immune responses in Aedes aegypti cells. PLoS One 5: e10678.
Dostert C, Jouanguy E, Irving P, Troxler L, Galiana-Arnoux D, Hetru C, Hoffmann JA, Imler JL, 2005. The Jak-STAT signaling pathway is required but not sufficient for the antiviral response of drosophila. Nat Immunol 6: 946–953.
Sabatier L, Jouanguy E, Dostert C, Zachary D, Dimarcq JL, Bulet P, Imler JL, 2003. Pherokine-2 and -3. Eur J Biochem 270: 3398–3407.
Mounsey A, Bauer P, Hope IA, 2002. Evidence suggesting that a fifth of annotated Caenorhabditis elegans genes may be pseudogenes. Genome Res 12: 770–775.
Platt KB, Linthicum KJ, Myint KS, Innis BL, Lerdthusnee K, Vaughn DW, 1997. Impact of dengue virus infection on feeding behavior of Aedes aegypti. Am J Trop Med Hyg 57: 119–125.
Salazar MI, Richardson JH, Sanchez-Vargas I, Olson KE, Beaty BJ, 2007. Dengue virus type 2: replication and tropisms in orally infected Aedes aegypti mosquitoes. BMC Microbiol 7: 9.
Lawson D, Arensburger P, Atkinson P, Besansky NJ, Bruggner RV, Butler R, Campbell KS, Christophides GK, Christley S, Dialynas E, Hammond M, Hill CA, Konopinski N, Lobo NF, MacCallum RM, Madey G, Megy K, Meyer J, Redmond S, Severson DW, Stinson EO, Topalis P, Birney E, Gelbart WM, Kafatos FC, Louis C, Collins FH, 2009. VectorBase: a data resource for invertebrate vector genomics. Nucleic Acids Res 37: D583–D587.
Calvo E, Tokumasu F, Marinotti O, Villeval JL, Ribeiro JM, Francischetti IM, 2007. Aegyptin, a novel mosquito salivary gland protein, specifically binds to collagen and prevents its interaction with platelet glycoprotein VI, integrin alpha2beta1, and von Willebrand factor. J Biol Chem 282: 26928–26938.
Rossingol PA, Spielman A, 1982. Fluid transport across the ducts of a mosquito. J Insect Physiol 28: 579–583.
Peng Z, Simons FE, 2007. Advances in mosquito allergy. Curr Opin Allergy Clin Immunol 7: 350–354.
Simons FE, Peng Z, 2001. Mosquito allergy: recombinant mosquito salivary antigens for new diagnostic tests. Int Arch Allergy Immunol 124: 403–405.
Sylvestre G, Gandini M, Maciel-de-Freitas R, 2013. Age-dependent effects of oral infection with dengue virus on Aedes aegypti (Diptera: Culicidae) feeding behavior, survival, oviposition success and fecundity. PLoS One 8: e59933.
Ramirez JL, Dimopoulos G, 2010. The Toll immune signaling pathway control conserved anti-dengue defenses across diverse Ae. aegypti strains and against multiple dengue virus serotypes. Dev Comp Immunol 34: 625–629.
Linthicum KJ, Platt K, Myint KS, Lerdthusnee K, Innis BL, Vaughn DW, 1996. Dengue 3 virus distribution in the mosquito Aedes aegypti: an immunocytochemical study. Med Vet Entomol 10: 87–92.
Mackenzie J, 2005. Wrapping things up about virus RNA replication. Traffic 6: 967–977.
Fernandez-Garcia MD, Mazzon M, Jacobs M, Amara A, 2009. Pathogenesis of flavivirus infections: using and abusing the host cell. Cell Host Microbe 5: 318–328.
Jin JP, Chong SM, 2010. Localization of the two tropomyosin-binding sites of troponin T. Arch Biochem Biophys 500: 144–150.
Ng ML, Hong SS, 1989. Flavivirus infection: essential ultrastructural changes and association of Kunjin virus NS3 protein with microtubules. Arch Virol 106: 103–120.
Lyles DS, 2000. Cytopathogenesis and inhibition of host gene expression by RNA viruses. Microbiol Mol Biol Rev 64: 709–724.
Hayes JD, Flanagan JU, Jowsey IR, 2005. Glutathione transferases. Annu Rev Pharmacol Toxicol 45: 51–88.
Sheehan D, Meade G, Foley VM, Dowd CA, 2001. Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochem J 360: 1–16.
Gui Z, Hou C, Liu T, Qin G, Li M, Jin B, 2009. Effects of insect viruses and pesticides on glutathione S-transferase activity and gene expression in Bombyx mori. J Econ Entomol 102: 1591–1598.
Drakesmith H, Prentice A, 2008. Viral infection and iron metabolism. Nat Rev Microbiol 6: 541–552.
Past two years | Past Year | Past 30 Days | |
---|---|---|---|
Abstract Views | 760 | 490 | 21 |
Full Text Views | 440 | 13 | 0 |
PDF Downloads | 201 | 15 | 0 |