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| Author(s): |
Thaís Camilo Corrêa
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Pirassununga. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Zootecnica e Engenharia de Alimentos (FZE/BT) |
| Defense date: | 2019-11-28 |
| Examining board members: |
Ricardo Luiz Moro de Sousa;
Mateus Maldonado Carriero;
Lara Borges Keid;
Hélio José Montassier;
Paulo Sérgio Monzani;
Cláudia Maris Ferreira Mostério
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| Advisor: | Ricardo Luiz Moro de Sousa |
| Abstract | |
Members of the genus Ranavirus, belonging to the family Iridoviridae, have been responsible for epizootics in ectothermic animals in various parts of the world, as they represent emerging pathogens capable of infecting three classes of vertebrates: bony fish, amphibians and reptiles. In Brazil, reports of Frog virus 3 (FV3)-associated ranavirus outbreaks have occurred since the mid-2000s, and are increasingly common mainly in commercial bullfrog farming. Therefore, ranavirus infections pose a risk to other aquaculture sectors, such as fish farming. In this context, aiming at the production of immunoreagents for use in diagnostic tests, the objectives were: to produce the recombinant MCP (Major capsid protein) gene from the Brazilian Ranavirus isolate, FV3-like; produce rabbit anti-rMCP polyclonal antibodies and, finally, standardize an ELISA-IB (Indirect-Blocking Enzyme Linked Immunosorbent Assay) test. To this end, the Rosetta (™)transformed plasmid pET28a / MCP (DE3) was constructed for the expression of the rMCP protein. Following purification, the production of polyclonal anti-rMCP antibodies was conducted in two young rabbits by three intramuscular and subcutaneous immunizations with the rMCP protein at 14-day intervals and the sera analyzed by Immunoblot. For standardization of the ELISA-IB test, bullfrog sera were sampled and experimental infection of Nile tilapia was carried out to obtain control sera. It is worth mentioning for the results, the successful expression of the 50kDa rMCP protein, however, in its insoluble form, under denaturing purification followed by dialysis. The rMCP triggered the production of polyclonal antibodies in rabbits as early as the second immunization. In ELISA-IB, rMCP protein was used as viral antigen, followed by sera tests and blocking antibodies (anti-rMCP polyclonal). However, problems with obtaining adequate control sera made the standardization of the test difficult, but it was possible to identify inhibition percentage in frog serum. The use of the produced immunoreagents and the ELISA-IB show promising potential for laboratory diagnosis of ranavirus infection in fish, as well as amphibians, in order to contribute to the improvement of sanitary conditions in Brazilian aquaculture. In addition, the obtained immunoreagents may provide further studies for the production of vaccine immunogens against Ranavirus. (AU) | |
| FAPESP's process: | 17/01356-4 - Immunobiological for the laboratory diagnosis of ranavirose: cloning and expression of the MCP gene of Brazilian Ranavirus isolate and production of polyclonal antibodies against recombinant MCP protein |
| Grantee: | Thaís Camilo Corrêa |
| Support Opportunities: | Scholarships in Brazil - Doctorate |