Establishment of a new bovine leukosis virus producing cell line.

Due to the prevalence of different bovine leukosis virus (BLV) species in the cattle population in Europe, problems may arise in the serological diagnosis of BLV infections. In addition, earlier investigations demonstrated that contamination of the BLV antigen-producing cell culture systems by bovine viral diarrhea virus (BVDV) may give rise to misinterpretation of serological test results after BVDV vaccination of cattle. By co-cultivation of peripheral leukocytes of a BLV-infected cow with a permanent sheep kidney cell line, a new BLV-producing cell line named PO714 was established. This line carries a BLV provirus of the Belgian species and has been tested to be free of a variety of possibly contaminating viruses and mycoplasms. Investigations of a panel of well-characterised sera by agar gel immunodiffusion (AGID) and capture ELISA (cELISA) tests using antigen prepared from this new cell line in comparison with antigen of the well-known cell line FLK/BLV yielded comparable results. False positive results caused by BVDV cross-reactions could be eliminated when tests were carried out with antigen derived from the new cell line.

Identification of different BLV provirus isolates by PCR, RFLPA and DNA sequencing.

A first attempt for the investigation of molecular epidemiology of BLV was carried out. PCR amplicons of a part of the env gene of BLV isolated from 309 cattle of different geographical origin were compared with known BLV env sequences. Using RFLPA most of the PCR products can be assigned to the Australian, the Japanese or the Belgian subgroup. A phylogenetic tree resulting from the comparison of the sequences of these env fragments demonstrates the relations and differences between and within the subgroups.

Involvement of intracellular Ca2+ in the regulation of bovine leukemia virus expression.

The calcium ionophore A23187, which was used to increase the intracellular calcium concentration ([Ca2+]i), was analyzed for effects on bovine leukemia virus (BLV) expression in two BLV infected cell lines. To clarify the role of intracellular free calcium in this response, [Ca2+]i was measured during ionophore treatment with the fluorescent calcium indicator Fura-2. Elevation of intracellular calcium under these conditions caused an enhancement of BLV gp51 and p24 synthesis as well as an activation of the BLV long terminal repeat (LTR) in a dose-dependent manner. Furthermore, it was observed that elevated levels of intracellular calcium following A23187 stimulation lead to activation of NF-kappaB. Based on inhibitor studies, we hypothesize that the effect of A23187 on BLV expression appears to be mediated by PKC.

A nucleotide deletion causing a translational stop in the protease reading frame of bovine leukaemia virus (BLV) results in modified protein expression and loss of infectivity.

Bovine leukaemia virus (BLV) is an oncogenic retrovirus that causes B-cell lymphocytosis and in the terminal stage of the disease lymphosarcoma. The comparison of the previously published BLV provirus sequence from Belgium, Australia and Japan showed that the protease gene (prt) of the Australian and the Japanese isolate contain a nucleotide deletion when compared to the Belgian isolate. Because all these proviruses were isolated from tumour tissue, the prt gene of functionally active and infectious proviruses from peripheral blood leucocytes (PBLs) of BLV-infected cattle and from BLV-infected fetal lamb kidney cells were sequenced. The only variations between these sequences and the Belgian isolate consist of nucleotide substitutions. The delection of one nucleotide of the prt gene of the Japanese and the Australian BLV tumour isolate caused a changed reading frame and a premature translational stop. It was shown that the Japanese provirus is non-infectious in transfected cell culture and in injected sheep. To analyse the impact of the prt mutation on viral protein expression and infectivity, the prt region of the Japanese provirus was exchanged with the prt region from the Belgian provirus. The resulting pBLVprtbelg was infectious in transfected cells and enabled the expression of gag and gag-precursor proteins. One sheep was injected with this mutated provirus and became positive in BLV-PCR, but no seroconversion was developed. The prt mutation of the Japanese tumour isolates was shown to be responsible for the loss of infectivity and changed viral expression. These results and the occurrence of this mutation in only two isolates from lymphosarcoma indicate a possible relation between the prt mutation and the induction of cell transformation.

[Serologic and virologic investigations on the presence of BLV infection in a dairy herd in Syria]. / Serologische und virologische Untersuchungen über das Vorkommen der BLV-Infektion in einer Milchviehherde in Syrien.

237 cattle of a dairy herd in Syria were tested for anti-BLV antibody by the ELISA. 194 animals were additionally examined by the agar gel immunodiffusions test (AGID) on BLV antibodies and 100 by polymerase chain reaction (PCR) for BLV provirus. BLV specific antibodies were determined by means of AGID and ELISA at 62.9% and 69.2% of the examined animals, respectively. Using the PCR method the BLV provirus was detected in 89% of the investigated cattle. Only one ELISA seropositive animal was negative for BLV provirus. The results show the high BLV contamination of this herd and lead to the presumption of wide spread enzootic bovine leukosis in Syria. In the case of the diagnosis of BLV-infection, the PCR-technique compared to the serological tests proved to be much more sensitive. By the detection of BLV antibody, the ELISA showed a higher sensitivity than the AGID and in this way, is advisable as a method of choice for screening investigations. Restriction enzyme and sequence analysis of PCR-amplificates demonstrate that different BLV provirus variants (A, B and C) in the examined herd occur, where the variant C which a high similarity to an Australian BLV provirus isolates showed, occurred most frequently at 92.5%.

[Possibilities and limitations for use of the polymerase chain reaction (PCR) in the diagnosis of bovine leukemia virus (BLV) infection in cattle]. / Möglichkeiten und Grenzen der Anwendung der Polymerase Kettenreaktion (PCR) bei der Diagnose der Infektion mit dem bovinen Leukosevirus (BLV) des Rindes.

Enzootic bovine leukosis is caused by the bovine leukemia virus (BLV) and has a world wide distribution in cattle. Due to the program for eradication of BLV-infections in Germany the BLV incidence in cattle declined and only few new cases seem to occur per year. On the other hand, BLV-infected cattle with low, transient or without BLV-antibody titers are difficult to identify as BLV-infected. These animals may be sources for new infections. It was the aim of this study to compare the suitability of agargel-immunodiffusion (AGID), enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) for diagnosis of BLV-infected cattle. We investigated a herd with 10 cows, where after a long period when the herd was negative suddenly a positive serological reaction appeared. In addition 64 animals from 6 federal states of different herds with doubtful serological reactions found in previous tests were included. In the herd with 10 cows we were able to detect BLV-infection in one animal 8 weeks earlier with PCR than with ELISA. Investigation of 56 adult cattle and 3 calves from different herds with both PCR and ELISA showed that 51 animals were positive in ELISA and 55 in PCR. Seven animal were positive in PCR and negative in ELISA. Three calves yielded negative results in PCR and positive results in ELISA. One cow which was positive in previous serological tests was negative in ELISA, AGID and PCR. Restriction fragment length polymorphism analysis demonstrated that the majority of the cattle was infected with the same BLV provirus variant. The four PCR variants used in this study yielded a similar sensitivity for BLV provirus detection. In conclusion, compared to the serological tests, PCR detects BLV-infection earlier in naturally infected cattle. The method is also a useful tool to exclude or confirm BLV-infection in cattle with doubtful serological results. PCR may be used to complement the serological tests in the diagnosis of BLV-infection.

[Polymerase chain reaction (PCR) for detection of BLV provirus-- a practical complement for BLV diagnosis?]. / Polymerase Kettenreaktion (PCR) zum Nachweis von BLV-Provirus--praktikable Ergänzung der BLV-Diagnostik?

A typical infection with bovine leukemia virus (BLV) induces a permanent antibody (Ab) response with high titers against BLV-antigens. In the last few years atypical courses of infection with low or transient BLV-Ab-titers or even lack of any detectable BLV-Ab-titers in animals with BLV-provirus integration have been described. This makes it difficult to eliminate BLV infection from herds using serological assays only. Whether or not polymerase chain reaction (PCR) is a useful tool to complement serological Ab-assays in BLV-eradication in herds was clarified in three ways: (i) different DNA-quick-preparations of blood were examined in nested PCR, (ii) cows of a BLV infected herd that was involved in a national eradication program were investigated for 6 months und (iii) BLV-provirus-variants occurring in this herd were differentiated. The results show, that even by using PCR it was not possible to detect all infected animals all the time and that eradication of BLV from this herd was not completed in this short time. The PCR is useful for the investigation of herds and more sensitive than ELISA. PCR using LTR-primers (34 positive cattle) was more sensitive than PCR with env-primers (30 positive cattle). Using PCR 34 BLV infected cattle were detected of which only 21 reacted in ELISA. Restriction enzyme analysis or sequence analysis of PCR-amplificates allowed the detection of virus variants and conclusions about the way of infection. PCR should be used for BLV-eradication in cattle herds with low BLV-incidence, for the investigation of new outbreaks or tumor cases in long term BLV free herds and for investigation of breeding cattle.

Provirus variants of the bovine leukemia virus and their relation to the serological status of naturally infected cattle.

Infection of cattle with the bovine leukemia virus (BLV) results in a strong permanent antibody response to the BLV antigens some weeks after infection. However, cattle may carry provirus and not have detectable antibody titers. To prove the occurrence of different BLV provirus variants in German cattle and to study the influence of special BLV variants on the immunoreaction, a 444-bp fragment of the env gene of 35 naturally BLV infected animals was analyzed. Seven different groups of BLV provirus variants were found on the basis of restriction fragment length polymorphism. Three BLV provirus variant groups and five additionally sequenced BLV isolates showed a high similarity to BLV provirus isolates from other geographical areas. The variation in nucleotide sequence of the five BLV isolates compared with nine previously sequenced BLV isolates ranged up to 5. 3%. While BLV provirus variant groups A, C, D, E, F, and G were clearly related to agar-gel immunodiffusion test (AGID)- and enzyme-linked immunosorbent assay (ELISA)-positive animals, BLV provirus variant group B was solely found in permanent AGID- and ELISA-negative or in transient ELISA-positive animals. Altogether, these results indicate that special BLV provirus variants may be responsible for atypical forms of BLV infection in cattle.

Evaluation of polymerase chain reaction (PCR) application in diagnosis of bovine leukaemia virus (BLV) infection in naturally infected cattle.

The practical application of polymerase chain reaction (PCR) for the diagnosis of bovine leukaemia virus (BLV) infections in naturally infected cattle was evaluated. Compared to serological tests the PCR was definitely found to be a more sensitive method, yielding the highest number of positive results (10% more compared to enzyme-linked immunosorbent assay, (ELISA), and 17.7% more compared to agar-gel immunodiffusion, (AGID)). In testing cattle from herds with BLV incidence under 5%, out of 52 provirus positive cattle only 43 were correctly identified by ELISA. When compared to AGID only 37 of the 52 PCR positive animals were correctly identified. Of 18 cattle imported from the Slovak Republic and kept in a quarantine stable, four were found to be BLV provirus positive by PCR, while serological tests indicated one animal positive and three negative. Therefore, it is impossible to prevent the spread of the infection from one country to another by serological testing only. Moreover, it is feasible to identify animals with changing antibody titres correctly by PCR. Using PCR we were also able to distinguish BLV infected from uninfected calves that were serologically positive due to colostral antibodies. Higher sensitivity of BLV provirus detection by PCR was achieved using env gene rather than tax gene specific primers. Negative results by PCR in cases of positive serological reactions are still possible, as shown in case of one adult animal. These findings indicate that PCR is a highly sensitive method and might be successfully used and economically advantageous for different practical applications in detection of BLV infection in naturally infected cattle.

Direct use of cell lysates in PCR-based diagnosis of bovine leukemia virus infection.

Polymerase chain reaction (PCR) has been used for direct detection of bovine leukemia virus (BLV) proviral DNA in cattle, but it is still mainly used for experimental research. One bottleneck for routine diagnosis of BLV by PCR has always been the isolation and purification of DNA. We compare the use of not purificated with highly-purified DNA in the PCR-based diagnosis of BLV infection. DNA extracted from whole blood by chloroform extraction (CP-DNA) and DNA prepared only by osmotic shock, washing, heating and freezing procedures (RPoS-DNA), were utilized. Fifteen cattle well characterized serologically were investigated for BLV-provirus with PCR using this different DNA preparations. With both methods all but one investigated animal were correctly identified. It was estimated that in case of CP-DNA PCR 10 BLV-provirus copies were sufficient to obtain a positive result. The sensitivity of RPoS-DNA PCR was similar. Because of the greater practicability of the latter technique we used it in a small field study with ten cattle. All serologically positive animals were correctly identified by the PCR. In addition one seronegative animal was found to carry BLV-provirus. Therefore RPoS-DNA PCR might be a good tool for the routine diagnosis of BLV-infected cattle.

Investigations of the Japanese bovine tumour virus (BLV)--its ability to express structural and regulatory BLV proteins.

The mechanism of BLV-induced tumorigenesis has not been clear up to now. Changes of viral protein expression in infected cells may be involved in the molecular events leading to BLV-induced leukaemogenesis. In this study Western blot investigations of cells transfected with plasmid DNA containing the complete Japanese BLV tumour clone provirus demonstrate that this provirus is unable to express gag and env proteins. Following this an attempt was made to express the genes from this provirus in eukaryotic and prokaryotic cells using the phagemid pBK-RSV (Stratagene), but not as fusion proteins. The protein patterns expressed from the 5' and the 3' region of the BLV genome were compared with those of FLK/BLV cells. The results indicate that there is a defect in this provirus located in the genome region between the gag and env gene.

Increase of antigen production in BLV-infected cell lines via additional expression of tax.

The selection of animals infected with the bovine leukaemia virus (BLV) is performed by the immunological detection of antibodies against the virus, commonly using the antigen gp51. Furthermore, research is being carried out to develop protective vaccines against BLV that have gp51 as their main component. Taking both of these factors into account, it is clear that there will be an increasing requirement for the virus antigen gp51 for some time to come. The permanently BLV-infected foetal lamb kidney cell line FLK/BLV (and its sublines) has been proved to be the most useful culture for the mass production of the virus antigen. Stable cell lines producing higher quantities of BLV antigen have not been established, either by subcloning of the FLK/BLV or by infection of other permanent cells with BLV. Here, a report is made on efforts to increase the expression of gp51 in BLV-infected cells via the additional expression of homologous transactivating virus protein tax. Selectable tax expression vectors that integrate into the host cell genome were constructed using BL provirus DNA fragments. Highly productive FLK/BLV cells were transfected with these vectors. Following selection with G 418, gp51-producing cell lines were established and tested for their productivity for several months. Some tax-vector-containing cell lines have produced 1.5-2 times more gp51 than the highly productive parental control cell line FLK/BLV 44-1.

[Different transactivation processes in two bovine leukemia virus producing cell lines]. / Unterschiedliches Transaktivierungsgeschehen in zwei bovines Leukämievirus produzierenden Zellinien.

Comparative studies were conducted through more than six months into quantitative of bovine leukaemia virus (BLV) antigen of FLC/BLV 44 and its FLC/BLV 44-4 subline by means of an enzyme immuno-assay (EIA), using monoclonal antibodies against gp51 and p24. Synthesis of gp51 (factors of two to six) and of p24 (factor of two) by FLC/BLV 44 was clearly higher than that by FLC/BLV 44-4. The transactivation status in either line was determined by transfer of the beta-galactosidase indicator organ under transcription control of BLV-LTR (in pBLV beta Gal plasmid). Transient experiments showed beta-galactosidase activity in the FLC/BLV 44 to be clearly higher than that in subline FLC/BLV 44-4. There is obviously in both cell lines a close correlation between intensity of BLV antigen synthesis and transactivation processes.

Isolation and characterization of prolyl hydroxylase from Chlamydomonas reinhardii.

Prolyl hydroxylase, which is responsible for the hydroxylation of peptidyl proline residues, has been isolated and purified from the green alga Chlamydomonas reinhardii. The enzyme, which appears to be loosely associated with microsomal membranes, was released into solution by sonication in the presence of detergent. Purification was achieved by ion-exchange chromatography followed by affinity chromatography using the immobilized substrate poly-L-proline. Apart from its differing substrate specificity the enzyme appears to possess similar molecular characteristics to prolyl hydroxylase isolated from animal tissues: the active enzyme is a tetramer of about 240-250 kDa and nonidentical monomers of 65 and 60 kDa. The monomers are capsule shaped having a dimension of 12×7 nm.