| Author(s): |
Helena Lage Ferreira
Total Authors: 1
|
| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 2007-12-02 |
| Examining board members: |
Clarice Weis Arns;
Tereza Cristina Cardoso;
Nelson Rodrigo da Silva Martins;
Fabio Trindade Maranhão Costa;
Maria Silvia Viccari Gatti
|
| Advisor: | Clarice Weis Arns; Renata Servan de Almeida |
| Field of knowledge: | Agronomical Sciences - Veterinary Medicine |
| Indexed in: | Base Acervus-UNICAMP; Biblioteca Digital da UNICAMP |
| Location: | Universidade Estadual de Campinas. Biblioteca Central Cesar Lattes; T/UNICAMP; F413e; Universidade Estadual de Campinas. Biblioteca do Instituto de Biologia; T/UNICAMP; F413e |
| Abstract | |
Avian metapneumovirus (AMPV) is the primary causative agent of severe rhinotracheitis in turkeys. AMPV belongs to the Paramyxoviridae family, Pneumovirinae subfamily, within the genus Metapneumovirus. It is associated with swollen head syndrome in chickens and is the source of significant economic losses to animal food production. The present study is divided in three parts. In the first part, the chicken embryo related (CER) cells beta-actin was evaluated. The CER beta actin gene was amplified by RT-PCR, and the amplicon was sequenced. The BHK21 and CER beta-actins were detected using hamster-specific primers. The results showed that such cells are closely related to BHK21 (p > 0.966), having a p-distance of 0.7 from chicken embryo fibroblasts. This confirms that CER cells are phylogenetically closely related to BHK21 cells. The second part of the study, we compared the specificity and detection limits of two newly designed conventional RT-PCRs (F and N genes) and two newly defined real time RT-PCR (RRT-PCR; (F and N genes), with an established RT-PCR (G gene) for AMPV detection. All the RT-PCR tested assays were able to detect the six isolates. The higher detection limits were observed at 10-5- fold and 10-5-fold dilutions of the N- and F- based RRT-PCR, respectively. Important to note that RRT-PCR assays generate fast and sensitive results, becoming a feasible alternative for virus isolation. In the third part, the silencing of AMPV by targeting its viral regions was promoted. We designed specific short interfering RNA (siRNA) targeting the nucleoprotein (N) and fusion (F) genes. Three days after the virus infection, the effect of siRNA in the virus replication was verified by virus titration, real time RT-PCR, and RT-PCR assays. AMPV titers presented reduction by 99.9%, when compared to the siRNA/F and siRNA/GFP treated samples. Also, real time RT-PCR results presented reduction of AMPV N, F and G mRNAs by 99.7%, when transfected with siRNA/N. Therefore, an siRNA sequence targeting the N gene was able to inhibit the AMPV production in vitro. In future studies, a combination of siRNAs targeting the RNA-polymerase complex may be used as a tool to study AMPV-infected cells or as an antiviral therapy (AU) | |
| FAPESP's process: | 03/14012-9 - The effect of RNAi application in avian pneumovirus infection |
| Grantee: | Helena Lage Ferreira |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |