Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays
Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized b...
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| Veröffentlicht in: | Journal of invertebrate pathology 2010-11, Vol.105 (3), p.312-321 |
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| creator | Jose, Seena Mohandas, A. Philip, Rosamma Bright Singh, I.S. |
| description | Immortal cell lines have not yet been reported from
Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1×), tryptose phosphate broth (2.95
g
l
−1), 20% FBS, N-phenylthiourea (0.2
mM), 0.06
μg
ml
−1 chloramphenicol, 100
μg
ml
−1 streptomycin and 100
IU
ml
−1 penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8
days. 5-Bromo-2′-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22
±
7% of cells at 24
h. Susceptibility of the cells to WSSV was confirmed by immunofluoresence assay using a monoclonal antibody against 28
kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT
50/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC
50. The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals. |
| doi_str_mv | 10.1016/j.jip.2010.08.006 |
| format | Article |
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Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1×), tryptose phosphate broth (2.95
g
l
−1), 20% FBS, N-phenylthiourea (0.2
mM), 0.06
μg
ml
−1 chloramphenicol, 100
μg
ml
−1 streptomycin and 100
IU
ml
−1 penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8
days. 5-Bromo-2′-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22
±
7% of cells at 24
h. Susceptibility of the cells to WSSV was confirmed by immunofluoresence assay using a monoclonal antibody against 28
kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT
50/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC
50. The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals.</description><identifier>ISSN: 0022-2011</identifier><identifier>EISSN: 1096-0805</identifier><identifier>DOI: 10.1016/j.jip.2010.08.006</identifier><identifier>PMID: 20807537</identifier><identifier>CODEN: JIVPAZ</identifier><language>eng</language><publisher>Amsterdam: Elsevier Inc</publisher><subject>Animal aquaculture ; Animal productions ; Animals ; Aquaculture - methods ; Biological and medical sciences ; BrdU assay ; Cell Culture Techniques - methods ; Cells, Cultured ; Crustacea ; Cytotoxicity ; Fluorescent Antibody Technique ; Fundamental and applied biological sciences. Psychology ; Gene Expression ; Genes, Viral ; Hemocytes - virology ; Immune-related genes ; Immunofluorescence ; Invertebrate aquaculture ; Invertebrates ; Models, Biological ; MTT assay ; Pathology ; Penaeidae - virology ; Penaeus monodon ; Primary hemocyte culture ; Reverse Transcriptase Polymerase Chain Reaction ; Virus Diseases - genetics ; Virus Diseases - immunology ; White spot syndrome virus ; White spot syndrome virus 1 - immunology ; White spot syndrome virus 1 - isolation & purification ; WSSV genes ; WSSV titration</subject><ispartof>Journal of invertebrate pathology, 2010-11, Vol.105 (3), p.312-321</ispartof><rights>2010 Elsevier Inc.</rights><rights>2015 INIST-CNRS</rights><rights>Copyright © 2010 Elsevier Inc. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c480t-5a90121c56d07197661d215e43e89f5f95b2e566f69f9ab1a759c4fe5c91ca693</citedby><cites>FETCH-LOGICAL-c480t-5a90121c56d07197661d215e43e89f5f95b2e566f69f9ab1a759c4fe5c91ca693</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.jip.2010.08.006$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3548,27922,27923,45993</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=23501383$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/20807537$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Jose, Seena</creatorcontrib><creatorcontrib>Mohandas, A.</creatorcontrib><creatorcontrib>Philip, Rosamma</creatorcontrib><creatorcontrib>Bright Singh, I.S.</creatorcontrib><title>Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays</title><title>Journal of invertebrate pathology</title><addtitle>J Invertebr Pathol</addtitle><description>Immortal cell lines have not yet been reported from
Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1×), tryptose phosphate broth (2.95
g
l
−1), 20% FBS, N-phenylthiourea (0.2
mM), 0.06
μg
ml
−1 chloramphenicol, 100
μg
ml
−1 streptomycin and 100
IU
ml
−1 penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8
days. 5-Bromo-2′-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22
±
7% of cells at 24
h. Susceptibility of the cells to WSSV was confirmed by immunofluoresence assay using a monoclonal antibody against 28
kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT
50/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC
50. The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals.</description><subject>Animal aquaculture</subject><subject>Animal productions</subject><subject>Animals</subject><subject>Aquaculture - methods</subject><subject>Biological and medical sciences</subject><subject>BrdU assay</subject><subject>Cell Culture Techniques - methods</subject><subject>Cells, Cultured</subject><subject>Crustacea</subject><subject>Cytotoxicity</subject><subject>Fluorescent Antibody Technique</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Expression</subject><subject>Genes, Viral</subject><subject>Hemocytes - virology</subject><subject>Immune-related genes</subject><subject>Immunofluorescence</subject><subject>Invertebrate aquaculture</subject><subject>Invertebrates</subject><subject>Models, Biological</subject><subject>MTT assay</subject><subject>Pathology</subject><subject>Penaeidae - virology</subject><subject>Penaeus monodon</subject><subject>Primary hemocyte culture</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>Virus Diseases - genetics</subject><subject>Virus Diseases - immunology</subject><subject>White spot syndrome virus</subject><subject>White spot syndrome virus 1 - immunology</subject><subject>White spot syndrome virus 1 - isolation & purification</subject><subject>WSSV genes</subject><subject>WSSV titration</subject><issn>0022-2011</issn><issn>1096-0805</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kc-OFCEQxonRuOPqA3gxXIwXe4TugW7iyWz8l2ziHvRMGChcJt0wAr1uv5JPaXVm1JsnqKrfV1Wpj5DnnG054_LNYXsIx23LMGbDljH5gGw4U7JhAxMPyYaxtm2wzC_Ik1IODH9CqsfkokWgF12_Ib9ucphMXugtTMkuFaidxzpnoMnTG4gG5kKnFJNLkZpCTaQh0rtQc8K0g5H6lOnP24DKckyVliW6nCZAJqO0ImlqSPH1mjAjNnA0TNMcgWYYTQVHvwMGcH_MUEpYxyCCq6Sa7oMNdcG5xSzlKXnkzVjg2fm9JN8-vP969am5_vLx89W768buBlYbYRTjLbdCOtZz1UvJXcsF7DoYlBdeiX0LQkovlVdmz00vlN15EFZxa6TqLsmrU99jTj9mKFVPoVgYRxMhzUXj6dqh5d2AJD-RNqdSMnh9PF1Tc6ZXh_RBo0N6dUizQaNDqHlx7j7vJ3B_FX8sQeDlGTDFmtFnE20o_7hOsHU2cm9PHOAt7gJkXWyAaMGFDLZql8J_1vgN90yyFQ</recordid><startdate>20101101</startdate><enddate>20101101</enddate><creator>Jose, Seena</creator><creator>Mohandas, A.</creator><creator>Philip, Rosamma</creator><creator>Bright Singh, I.S.</creator><general>Elsevier Inc</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7U9</scope><scope>8FD</scope><scope>F1W</scope><scope>FR3</scope><scope>H94</scope><scope>H95</scope><scope>H98</scope><scope>L.G</scope><scope>P64</scope><scope>RC3</scope></search><sort><creationdate>20101101</creationdate><title>Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays</title><author>Jose, Seena ; Mohandas, A. ; Philip, Rosamma ; Bright Singh, I.S.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c480t-5a90121c56d07197661d215e43e89f5f95b2e566f69f9ab1a759c4fe5c91ca693</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>Animal aquaculture</topic><topic>Animal productions</topic><topic>Animals</topic><topic>Aquaculture - methods</topic><topic>Biological and medical sciences</topic><topic>BrdU assay</topic><topic>Cell Culture Techniques - methods</topic><topic>Cells, Cultured</topic><topic>Crustacea</topic><topic>Cytotoxicity</topic><topic>Fluorescent Antibody Technique</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Expression</topic><topic>Genes, Viral</topic><topic>Hemocytes - virology</topic><topic>Immune-related genes</topic><topic>Immunofluorescence</topic><topic>Invertebrate aquaculture</topic><topic>Invertebrates</topic><topic>Models, Biological</topic><topic>MTT assay</topic><topic>Pathology</topic><topic>Penaeidae - virology</topic><topic>Penaeus monodon</topic><topic>Primary hemocyte culture</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>Virus Diseases - genetics</topic><topic>Virus Diseases - immunology</topic><topic>White spot syndrome virus</topic><topic>White spot syndrome virus 1 - immunology</topic><topic>White spot syndrome virus 1 - isolation & purification</topic><topic>WSSV genes</topic><topic>WSSV titration</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Jose, Seena</creatorcontrib><creatorcontrib>Mohandas, A.</creatorcontrib><creatorcontrib>Philip, Rosamma</creatorcontrib><creatorcontrib>Bright Singh, I.S.</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>ASFA: Aquatic Sciences and Fisheries Abstracts</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) 1: Biological Sciences & Living Resources</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) Aquaculture Abstracts</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) Professional</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><jtitle>Journal of invertebrate pathology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Jose, Seena</au><au>Mohandas, A.</au><au>Philip, Rosamma</au><au>Bright Singh, I.S.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays</atitle><jtitle>Journal of invertebrate pathology</jtitle><addtitle>J Invertebr Pathol</addtitle><date>2010-11-01</date><risdate>2010</risdate><volume>105</volume><issue>3</issue><spage>312</spage><epage>321</epage><pages>312-321</pages><issn>0022-2011</issn><eissn>1096-0805</eissn><coden>JIVPAZ</coden><abstract>Immortal cell lines have not yet been reported from
Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1×), tryptose phosphate broth (2.95
g
l
−1), 20% FBS, N-phenylthiourea (0.2
mM), 0.06
μg
ml
−1 chloramphenicol, 100
μg
ml
−1 streptomycin and 100
IU
ml
−1 penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8
days. 5-Bromo-2′-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22
±
7% of cells at 24
h. Susceptibility of the cells to WSSV was confirmed by immunofluoresence assay using a monoclonal antibody against 28
kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT
50/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC
50. The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals.</abstract><cop>Amsterdam</cop><pub>Elsevier Inc</pub><pmid>20807537</pmid><doi>10.1016/j.jip.2010.08.006</doi><tpages>10</tpages></addata></record> |
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| issn | 0022-2011 1096-0805 |
| language | eng |
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| source | MEDLINE; Elsevier ScienceDirect Journals Complete |
| subjects | Animal aquaculture Animal productions Animals Aquaculture - methods Biological and medical sciences BrdU assay Cell Culture Techniques - methods Cells, Cultured Crustacea Cytotoxicity Fluorescent Antibody Technique Fundamental and applied biological sciences. Psychology Gene Expression Genes, Viral Hemocytes - virology Immune-related genes Immunofluorescence Invertebrate aquaculture Invertebrates Models, Biological MTT assay Pathology Penaeidae - virology Penaeus monodon Primary hemocyte culture Reverse Transcriptase Polymerase Chain Reaction Virus Diseases - genetics Virus Diseases - immunology White spot syndrome virus White spot syndrome virus 1 - immunology White spot syndrome virus 1 - isolation & purification WSSV genes WSSV titration |
| title | Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays |
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