Ester Teresa González

Provirus variants of bovine leukemia virus in naturally infected cattle from Argentina and Japan

A serologic subgroup of bovine leukemia virus (BLV) has not been identified, whereas genetic diversity among BLVs has been reported by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). To investigate the distribution of BLV provirus variants, 42 isolates from Argentina and Japan were examined by nested PCR for a segment of the env gene, followed by DNA sequencing. The nucleotide sequences were compared with other previously characterized BLV variants from different geographical areas (Belgium, France, Italy, North America, Australia, Japan and Argentina). The majority of analyzed segments had a tendency for nucleotide substitution without changing the amino acid. The constructed phylogenetic tree showed the relations and differences between proviruses and within each one. Most of the samples in Argentina formed one cluster. The samples in Japan, except one, also formed one cluster and some of them showed high homology with the isolates from Australia and the USA. Considering the sequence analysis of env PCR products of all Japanese and Argentine samples and comparing them with the other previously isolated sequences, the variation was up to 3.5% and was characterized geographically in each area.

Genetic heterogeneity among bovine leukemia virus genotypes and its relation to humoral responses in hosts

The existence of bovine leukemia virus (BLV) genotypes was investigated by restriction fragment length polymorphism (RFLP) analysis using bovine peripheral blood leukocytes collected from different geographical areas of Japan. For this purpose a nested polymerase chain reaction (PCR) for a 444 bp fragment of the envelope (env) gene was used because it was previously reported that this region might be responsible for the serological status in the host. The PCR products from 60 samples of BLV-infected cells were digested with endonucleases BamH I, Bgl I, Bcl I, Hae III and Pvu II. RFLP analysis demonstrated that there were six different genotypes of BLV present among cattle in Japan. In some herds PCR-positive animals were infected with only one genotype, but in other herds a few genotypes were found. One genotype was dominant throughout infected cattle and it was also detected in neoplastic cells from three of four animals with lymphosarcoma and three cell lines persistently infected with BLV. Production of antibodies to BLV in each cattle was surveyed by agar gel immunodiffusion and indirect hemagglutination tests, and the results were compared with those obtained from PCR. No genotype related to decreased immunoreactivity was detected. The difference in anti-viral immune responses of each animal appears to be related to the infection stage and other host factors, not to genetic heterogeneity of the envelope gene.

A rapid and sensitive diagnosis of bovine leukaemia virus infection using the nested shuttle polymerase chain reaction

Bovine leukaemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). In Argentina, where a program to eradicate EBL has been introduced, sensitive and reliable diagnosis has attained high priority. Although the importance of the agar gel immunodiffusion test remains unchanged for routine work, an additional diagnostic technique is necessary to confirm cases of sera with equivocal results or of calves carrying maternal antibodies.Utilizing a nested shuttle polymerase chain reaction, the proviral DNA was detected from cows experimentally infected with as little as 5 ml of whole blood from BLV seropositive cows that were nonetheless normal in haematological terms. It proved to be a very sensitive technique, since it rapidly revealed the presence of the provirus, frequently at 2 weeks postinoculation and using a two-round procedure of nested PCR taking only 3 hours. Additionally, the primers used flanked a portion of the viral genome often employed to differentiate BLV type applying BamHI digestion. It is concluded that this method might offer a highly promising diagnostic tool for BLV infection.

Enzootic bovine Leukosis: development of an indirect enzyme linked immunosorbent assay (I-Elisa) in seroepidemiological studies

Bovine Leukemia Virus (BLV) is the etiologic agent of Enzootic Bovine Leukosis, a retrovirus exogenous to the bovine species. Once infected, there is no detectable viraemia but instead there is a strong and persistent immunological response to BLV structural proteins, essentially the gp51 envelope glycoprotein and the mayor core protein p24. We describe the test procedure of an indirect ELISA (I-ELISA) using polyclonal reagents for the detection of antibodies to BLV. For comparison, the sera were simultaneously tested by agar gel immunodiffussion (AGID) test, which is currently used as diagnostic standard for BLV infection. The antigen applied does not require a high degree of purification and the data from the analysis of the negative sera showed that the establishment of a cut-off level corresponding to 3 times the standard deviation (SD) above the mean for the negative control set of sera provided acceptable specificity, reducing the risk of false positives results. A comparison of the results obtained by AGID test and I-ELISA showed that considering a total of 465 serum samples, all of the 234 samples (50%) that were positive by AGID were positive to the I-ELISA. Of 225 serum samples which yielded negative results in the AGID test, 69 (15%) were found to be positive by the I-ELISA and 156 (33%) were negative by both techniques. Few sera (2%) that were non-specific by AGID were defined as negative or positive by I-ELISA.

Expression of p24 gag Protein of Bovine Leukemia Virus in Insect Cells and Its Use in Immunodetection of the Disease

Bovine leukemia is a common retroviral infection of cattle. The disease is characterized by a strong immunological response to several viral proteins, but the antibodies against p24 and gp51 are predominant. In this study, a recombinant baculovirus containing the gag gene p24 was constructed and the protein, used as antigen, analyzed by western blot and an indirect in-house rp24-ELISA test. This allowed detecting the presence of antibodies for bovine leukemia virus in a panel of cattle sera. The authentication of the protein expands its potential use for different medical applications, from improved diagnosis of the disease to source of antigens to be included in a subunit vaccine.

Analysis of the pX region of bovine leukemia virus in different clinical stages of Enzootic Bovine Leukemia in Argentine Holstein cattle.

Bovine leukemia virus (BLV) infection in cattle causes Enzootic Bovine Leukemia (EBL). About 30% of infected cattle develop persistent lymphocytosis (PL), a 0.1-5% develops tumors, and a 70% remains asymptomatic in an aleukemic stage (AL). Regulatory genes of BLV (Tax, Rex, R3 and G4) are located in a region known as pX(BLV). The variability of those genes had been postulated with the progression of the disease. The aim of this work was to compare the wild-type proviral pX(BLV) region at different stages of BLV natural infected cattle from Argentine Holstein. Pairs of primers were designed to amplify the proviral pX region of 12 cattle by PCR, and products were then sequenced, aligned and compared both with each other and with the reference sequence. Results show a divergence percentage from 0 to 6.1 for the Tax gene, from 0 to 9.4% for the Rex gene, from 0 to 12.1% for the R3 gene and finally from 0 to 6.5% for the G4 gene. Results obtained with hierarchical clustering showed two clusters well differentiated, where the members of each cluster are cattle that had tumor, PL and AL, not allowing differentiate those two cluster by clinical stage.

Presence of Gumprecht shadows (smudge cells) in bovine leukemia virus-positive cattle.

Enzootic Bovine Leukosis is a chronic disease caused by the bovine leukemia virus (BLV). Smudge cells, also known as Gumprecht shadows, are not simple artifacts of slide preparation, but ragged lymphoid cells found mainly in peripheral blood smears from human patients with chronic lymphocytic leukemia. In this study, we report the presence of Gumprecht shadows in peripheral blood from BLV-positive cattle.