Genomanalyse verschiedener Isolate des Bovinen Leukosevirus unter besonderer Berücksichtigung des serologischen Status und der geographischen Herkunft des Rindes

Abstract

For decades serological methods seemed to be useful and sufficient to diagnose BLV infection in cattle. Repeatedly it was found out, that antigen variants and outcoming antibody (AK)-serotypes do not appear. The occurence of serological negative provirus carrying cattle and the lack of BLV-provirus in Enzym-Linked-Immunosorbent Assay (ELISA)- and Agargel-Immunodiffusion-Test (AGID)-positive animals lead to the assumption, that with the selection of BLV free herds specific mutants of provirus dominated. For this reason the aim of the paper was to investigate the nucleotid sequence of different BLV isolates and the resulting aminoacid sequence in consideration of the serological state and geograpical origin of the infected cattle. Simultaneously the influence of nucleid- and aminoacid-changes on the RNA- and protein-secundary structure should be investigated. Especially in the veterinary medicine DNA sequence analysis is limited because it is much timeconsuming and expensive. For this reaseon different sequencing methods should be compared with respect to their practicability and rentability. 1. In comparison of different "Sanger principle" using sequencing methods (method after Sommer, Hot Tub sequencing, T7-Sequencing and Auto Read sequencing) with respect to practicability and rentability the T7-sequencing method was the most suitable. With a minimum of 1,5 - 2,0 µg ds DNA up to 450 nucleotides could be read during 4 h from a vertical, wedge-shaped gel. 2. The direct sequencing of polymerase chain reaction (PCR) products in comparision with the sequencing of cloned PCR products has various advantages: less time consuming, high purity of DNA template and less danger of sequencing DNA fragments with PCR induced mutationes. The quantity of detectable nucleotides sequencing PCR products synthesized in single- or double PCR with a maximum of 230 bp was lower than sequencing cloned PCR products. Using fragments from asymmetric PCR´s the number of detectable nucleotides were raised to 400 bp. 3. Investigations for the detection of BLV provirus in 37 cattle of different herds with ELISA, AGID, leucocyte short term culture and PCR enabled a division of the probands into four groups:

* 10 animals (27%) serology positive, carrier of provirus; group 1 * 3 animals (8%) serology uncertain, carrier of provirus; group 2 * 10 animals (27%) serology negative, carrier of provirus; group 3 * 14 animals (38%) serology negative, no carrier of provirus group 4 4. The comparison of the diagnostic methods AGID, ELISA, leucocyte short term culture and PCR showed, that the ELISA is not sufficient to detecte all provirus carrier. The PCR is necessary for the detection of cattle with weak serological signals in BLV eradication. 5. The sequencing of the BLV long terminal repeat (LTR) region from FLK (fetal lamb kidney) -BLV provirus is highly comperable with the Japanese isolat (Sagata et al., 1985a). Two detected nucleitid substitutiones found in this region were in structural less significant regions of the sequence. 6. The DNA sequencing of parts from the envelope (env)- polymerase- and Tax protein (tax)-region of BLV indicate the existence of three groups of mutants from BLV:

* BLV-D+ (BLV mutant of serology positive, carrier of provirus) * BLV-D+/- (BLV mutant of serology instable, carrier of provirus) * BLV-D- (BLV mutant of serology negative, no carrier of provirus) 7. For the occurence of serologic negative cattle there are at least four reasonable explanationes:

* congenital immuntolerance following intrauterin infection * BLV provirus detection in the early stage of BLV infection, before detectable antibody titers have developed * selection of virus mutants whith weak immunogenicity (BLV virus selection hypothesis) * selection of cattle with weakly immune response by parallel working with different eradication concepts (BLV host selection hypothesis) 8. Changes in nucleotid sequence influence the RNA- and protein secundary structure. Potentiell splice donors and acceptors are mainly conservative. BLV isolates from some seropositive cattle were lacking the gp51 sequence epitop G. 9. The result of the study indicate that there is a relation between the serological state of the infected cattle and the DNA sequence of the BLV mutant but not between BLV provirus mutant and the geographical origion of the host. 10. Partial DNA sequencing of the tax\- and env-region of the BLV from different FLK-sublines show, that there are differences beetween the FLK-sublines as well as between other BLV isolates. This difference indicates an antigenic drift between BLV proviruses that may influence the suitability of FLK-BLV antigenes in test systems for diagnosis of bovine leukosis infection.