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Ass. Lect. Marawan Adel Marawan Basuony :: Theses :

Title Molecular studies on Bovine leukemia Virus
Type PhD
Supervisors Prof. Abdel-moneim M. Moustafa, Prof. Faysal K. Arnaout, Prof. El-Sayed M. Galila and Prof. Junzo Norimine
Year 2017
Abstract Enzootic bovine leucosis (EBL) is a disease caused by bovine leukemia virus (BLV) which is RNA virus, belongs to the family Retroviridae, genus Deltaretrovirus with structure and function relate to the Human T lymphotropic virus I and II (HTLV I and II). EBL has worldwide distribution and can result in economic losses for cattle industry (Rodriguez et al., 2009; Murakami et al., 2011; Juliarena et al., 2013; Gutiérrez et al., 2014 and Nekouei et al., 2015). BLV infection is transmitted mainly by horizontal routes but also may be transmitted vertically by ingestion of colostrum or in utero infection)Gillet et al., 2013; Bartlett et al., 2014; Şevik et al., 2015 and Mekata et al., 2015a). Most BLV-infected cattle (about 70 %) do not show any clinical symptoms, and about 30 % of them develop persistent lymphocytosis (PL) and 1-5 % of the infected cattle develop malignant B-cell lymphosarcomas causing EBL (Balic et al., 2012; Kim et al., 2015, Ohno et al., 2015 and Pandey et al., 2017). BLV is encoded by gag, pro, pol and env essential genes which are important for the production of infectious virions, and are flanked by 2 identical long terminal repeats (LTRs) like others retroviruses (Alkan et al., 2011; Aida et al., 2013; Juliarena et al., 2013 and Frie and Coussens, 2015). The BLV envelope gene (env) encodes one polyprotein precursor (gpr72) which is cleaved into gp51 surface glycoproteins and gp30 transmembrane glycoprotein. It plays an essential role in the viral life cycle and infection and is the target of the neutralizing antibodies (Balic et al., 2012 and De Brogniez et al., 2015). Exosomes are small (50–90 nm) cup shape membrane nanovesicles of endocytic origin that are released into the extracellular environment through fusion of multivesicular bodies (MVB) with the plasma membrane (Van Niel et al., 2006; Conde-Vancells et al., 2008; Zomer et al., 2010 and Phinney and Pittenger, 2017). It is released by almost all cell types including those of hematological origin, such as B-cells and have also been confirmed in all bodily fluids such as bronchoalveolar fluid, cerebro-spinal fluid, blood, urine, saliva, amniotic fluids, semen, and synovial fluid (Zech et al., 2012 and Sharma et al., 2016). Exosomes play an important role in cell-to-cell communication and influence both physiological and pathological processes with a broad range of functions, depending on their cell or tissue of origin and play an important role in the whole process of tumor metastasis pathologically (An et al., 2015; Lin et al., 2015 and Kouwaki et al., 2017). Enzyme Linked Immunosorbent Assay (ELISA) as a serological diagnostic technique has been used more extensively to identify BLV-infected cattle worldwide with higher sensitivity and specificity (Jimba et al., 2012, Kobayashi et al., 2014 and Buehring, 2017). PCR and real time PCR are recommended for diagnosis of BLV as it provide the highest sensitivity besides, convention PCR can be also followed by sequence and phylogenetic analysis to see the distribution of various BLV genotypes worldwide (Lojkić et al., 2013; Polat et al., 2015; Takeshima et al., 2015 and Takeshima et al., 2016). A phylogenetic studies of BLV env gene from strains isolated worldwide demonstrated that the virus can be divided into ten genotypes from one to 10 (Vafin et al., 2014; Kim et al., 2015; Lee et al., 2016, Polat et al., 2016 and Pluta et al., 2017) and these genotypes are correlated with their geographic origin (Rodriguez et al., 2009 and Polat et al., 2015). The molecular constituents in exosomes (including exosomal RNAs) have been found to be associated with certain diseases and treatment responses, indicating that it serves as a noninvasive diagnostic tool (Boyiadzis and Whiteside, 2015; Lin et al., 2015, Sharma et al., 2016 and Kouwaki et al., 2017). Therefore our objectives were: 1. Antibodies detection of BLV infection in plasma samples collected from cattle farms in different localities of Miyazaki prefecture (Northern, central and southern), Japan using ELISA. 2. Molecular detection of BLV proviral DNA (BLV gp51 env gene) using PCR and BLV proviral load using real-time PCR in the whole blood of positive samples. 3. Investigation of the genetic relatedness among the examined BLV strains worldwide through sequencing and phylogenetic analysis of BLV env gene. 4. Determination the role of exosomes in extracellular delivery of BLV genetic materials and proteins derived from infected cells through detection of exosomal BLV- RNA by RT-PCR and env protein by western blot.
University Benha
Country Egypt
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