Detection and molecular characterization of bovine leukemia viruses from Jordan

Bovine leukemia virus (BLV) is distributed worldwide. BLV has many effects on the health status and productivity of infected animals and is a potential risk for humans. In this study, we aimed to investigate the presence of and genotype bovine leukemia viruses on Jordanian dairy farms. Nested PCR coupled with RFLP and direct sequencing of a partial fragment of the env gene were carried out. Two BLV genotypes were found, genotypes 1 and 6. These genotypes were identified by nested PCR-RFLP of 444 bp of the env gene by restriction digestion with HaeIII, Bcl I and Pvu II. However, BLV-Jordan-10 seems to represent an entirely new genotype in our phylogenetic analysis. The nucleotide sequence identity between these two Jordanian BLV genotypes (1 and 6) was 96.2?%. The nucleotide sequence identity between Jordanian BLV genotype 1 and other reference BLV genotype 1 strains ranged from 99?% to 99.5?%. The nucleotide sequence similarity of the Jordanian BLV genotype 6 to other BLV genotypes ranged from 90?% to 96.7?%. A neutralizing motif and CD8⁺ T-cell epitope were found in the env protein of both Jordanian isolates. In this study, we documented the presence of two BLV genotypes (1 and 6) on Jordanian dairy farms.     

Detection of bovine leukemia virus in cattle by the polymerase chain reaction.

Bovine leukemia virus (BLV) is widely distributed in U.S. cattle herds. It infects B lymphocytes and causes neoplastic disease in 5-10% of infected animals. Direct economic losses are incurred as a result of death, reduced milk production and condemnation at slaughter. Thus the identification of cattle infected with BLV is of significant concern to the U.S. cattle industry. For this reason, polymerase chain reaction (PCR) amplification was used to examine seropositive and seronegative cattle for the presence of BLV DNA in peripheral blood mononuclear cells. Using an amplification protocol able to detect 1 viral genome in 100,000 cells, BLV was not detected in 7 seronegative cattle in an infected herd. BLV sequences were detected in 13 of 18 seropositive animals with various levels of infection as determined by in vitro lymphocyte culture and electron microscopy. An active infection was demonstrated in one animal, based on the presence of viral RNA. These findings indicate that PCR is a sensitive method for the detection of BLV in cattle and provides new information regarding the dynamics of the infection.     

Detection of bovine leukemia virus in tissue cultures

Syncytium formation was demonstrated in cocultivation of cow and sheep embryo kidney cells with peripheral blood lymphocytes from leukemic cattle. The beginning of this phenomenon was observed at 3--4, and maximum development at 6--8 days of cocultivation. Lymphocytes of 22 out of 24 leukemic animals were capable of inducing syncytium formation. Parallel examinations of cocultivated cells by the direct immunofluorescence procedure using fluorescent antibodies from sera of leukemic cows gave positive results in 23 our of 24 cases, whereas lymphocytes of 12 normal cows never induced either syncytium or specific antigens.     

Cytotoxic antibody in cattle and sheep exposed to bovine leukemia virus

Summary Sera from bovine leukemia virus-infected cattle and sheep lysed fetal lamb kidney cells in the presence of rabbit complement. This cytolytic activity was removed completely from the sera by absorption with bovine leukemia virus (BLV). Antiserum against surface glycoprotein antigens of BLV contained cytolytic antibody but antiserum against the internal protein p24 did not. The complement-dependent antibody cytotoxicity test employing the trypan-blue dye exclusion method appeared to be suitable for routine diagnosis of BLV infection.With 2 Figures     

Cellular basis of persistent lymphocytosis in cattle infected with bovine leukemia virus.

Peripheral blood lymphocytes from 14 cattle infected with the bovine leukemia virus (BLV) and 14 BLV-free cattle were examined by the membrane immunofluorescent antibody technique to detect surface immunoglobulin (S-Ig) and by the erythrocyte-antibody-complement (EAC) rosette test for the detection of complement receptors. Direct comparisons of the percentages of S-Ig-bearing cells and EAC rosette-forming cells in both infected and BLV-free animals showed no evidence for the presence of a substantial population bearing one surface marker but not the other. The data showed that cells with surface markers characteristic of B lymphocytes are responsible for most of the increase in peripheral blood lymphocytes which may accompany BLV infection. The release of infectious BLV and the spontaneous uptake of thymidine by short-term cultured peripheral blood lymphocytes from BLV-infected cattle were also studied. The results indicate that both of these activities are function of B lymphocytes.     

Comparison of the copy numbers of bovine leukemia virus in the lymph nodes of cattle with enzootic bovine leukosis and cattle with latent infection

To establish a diagnostic index for predicting enzootic bovine leukosis (EBL), proviral bovine leukemia virus (BLV) copies in whole blood, lymph nodes and spleen were examined by quantitative real-time PCR (qPCR). Cattle were divided into two groups, EBL and BLV-infected, based on meat inspection data. The number of BLV copies in all specimens of EBL cattle was significantly higher than those of BLV-infected cattle (p < 0.0001), and the number of BLV copies in the lymph nodes was particularly large. Over 70 % of the superficial cervical, medial iliac and jejunal lymph nodes from EBL cattle had more than 1,000 copies/10 ng DNA, whereas lymph nodes from BLV-infected cattle did not. These findings suggest that the cattle harboring more than 1,000 BLV copies may be diagnosed with EBL.     

Identification and molecular characterization of a bovine G3 rotavirus which causes age-independent diarrhea in cattle

G3 rotaviruses have been reported rarely in cattle, and none have been characterized. We report the first genomic characterization of a bovine G3 rotavirus, CP-1, which had been biologically characterized in vivo and shown to cause age-independent diarrhea. CP-1 was a G3 rotavirus as its VP7 had 92 to 96% deduced amino acid identity to those of G3 rotaviruses. However, initially, CP-1 was identified as a G10 rotavirus by RT-PCR even though the CP-1 VP7 had only 81 to 85% deduced amino acid identity to those of G10 rotaviruses. Rotavirus CP-1 was of P[5] specificity, a type common in cattle, and had a bovine NSP1 and NSP4. These results added another animal species to those in which G3 rotaviruses have been found, characterized a bovine rotavirus which caused age-independent diarrhea in calves, and raised the possibility that bovine G3 rotaviruses may be misdiagnosed as G10 rotaviruses.     

Properties of density gradient-fractionated peripheral blood leukocytes from cattle infected with bovine leukemia virus.

Discontinuous bovine serum albumin gradients were used to fractionate peripheral blood leukocytes from bovine leukemia virus (BLV)-free and BLV-infected cows. The release of infectious BLV and spontaneous incorporation of [3H]thymidine were not properties of density gradient-fractionated leukocytes from a BLV-free cow. When leukocytes from BLV-infected cattle were fractionated, B lymphocytes which spontaneously incorporated [3H]thymidine could be separated as a distinct subpopulation from B lymphocytes which replicated infectious BLV. Density gradient fractionation of leukocytes from a cow with lymphosarcoma is also reported. A fall in lymphocyte count at the time of tumor development is attributed to the loss of B lymphocytes which spontaneously incorporate [3H]thymidine.     

Morphogenesis of bovine leukemia virus.

The morphogenesis of bovine leukemia virus (BLV) was studied in short-term cultures of leukocytes from cows with persistent lymphocytosis and in BLV-producing cell lines. Few budding particles were found. They consisted of one shell underneath the cell membrane with granules attached to the inner side. When the shell is completed the budding particle could follow two pathways: It could (a) bud from the cell membrane to give rise to a free immature particle or (b) mature while still in contact with the cell, by condensation of the shell and the granules into a nucleoid, and subsequently bud from the cell membrane as a mature virion. A different pathway of morphogenesis, probably followed by the majority of the virions, is proposed based on the following observations: (1) Low numbers of budding particles on the cell membrane, (2) condensation of electrondense material within the cytoplasm resembling virus particles in the first stage of budding, and (3) immature and mature particles lying free in the cytoplasm. This pathway of morphogenesis suppose the formation of immature and mature particles within the cytoplasm without a budding process. Immunoferritin studies on these BLV-producing cells using bovine and goat anti-BLV sera have shown labeling of the BLV particles. The cell surface, however, was labeled only rarely and then in small areas. This means that on the cell surface of BLV-producing cells very few viral structural polypeptides are present. A comparison of the morphology of BLV and several other oncornaviruses leads to the conclusion that the morphogenesis of BLV is different from that of the type B and C and other similar particles such as Mason-Pfizer monkey virus and bromodeoxyuridine-induced guinea pig leukemia virus.     

Immunological characterization of a low molecular weight polypeptide of murine leukemia virus

A low molecular weight polypeptide (MW 16,000) of the Rauscher strain of mouse leukemia virus (MuLV) has been isolated. A very sensitive and highly specific radioimmunoassay has been developed for its quantitation. The present studies indicate that this polypeptide is virus-coded and antigenically distinct from another virion protein, the group-specific (gs) antigen Different strains of MuLV contain antigens immunologically cross-reactive with the low molecular weight polypeptide. Studies are presented comparing the level of expression of this virion antigen in normal and transformed cells.     

Detection of the provirus of the bovine leukemia virus in bovine peripheral blood leukocytes by means of DNA hybridization

The method allowing the detection of BLV proviral DNA in the peripheral blood leukocytes of cattle is reported. Cell DNA from leukocytes used without preliminary cultivation was dot-hydridized with 32P-labeled plasmid that included a fragment of BLV proviral DNA. In parallel, sera from the cattle under study were tested by immunodiffusion assay (ID). The results indicate that dot-hybridization assay is more sensitive as a diagnostic test than ID because it detects BLV infection in apparently normal cattle which was seronegative by ID.     

Genetic characterization of a noncytopathic bovine viral diarrhea virus 2b isolated from cattle in China

In January 2013, several clinical signs of cattle with diarrhea, cough, nasal discharge, and fever were reported in Jilin province, China. One virus named SD1301 was isolated and identified. Complete genome of the virus is 12258nt in length and contains a 5′UTR, one open reading frame encoding a polyprotein of 3,897 amino acids and a 3′UTR. Phylogenetic analysis of 5′UTR, N^pro, E1 and E2 gene demonstrated the virus belonged to BVDV 2b, and genetically related to the BVDV strain Hokudai-Lab/09 from Japan in 2010. This bovine viral diarrhea virus displays a unique genetic signature with 27-nucleotide deletion in the 5′UTR, which is similar to the bovine viral diarrhea virus C413 (AF002227). This was the first confirmed isolation of ncp BVDV2b circulating in bovine herd of China.     

Structural and functional characterization of mutants of the bovine leukemia virus transactivator protein p34

Mutants of the bovine leukemia virus (BLV) transactivator protein (tat, tax, p34, the XLOR gene product) were constructed by site-directed deletions, in-phase linker insertions, or fragment replacements (swapping) among BLV variants. The mutant constructs were transfected into cos cells and transiently expressed. Western blot analysis using a mixture of monoclonal antibodies to wild-type p34 revealed the presence of mutated XLOR gene products in all the mutants tested. The transactivating activity of 11 tax mutants containing site-directed deletions and in-phase linker insertions was completely abolished. Only the swapping mutant tested, a hybrid between two BLV variants, transactivated LTR-directed gene expression at wild-type levels. These data illustrate the narrow range of structural variations that allow full activity of the BLV tax product and suggest that the present molecular structure of the transactivator protein results from heavy evolutionary constraints.