Marc Mammerickx

Bovine Leukemia Virus Involvement In Enzootic Bovine Leukosis

Publisher Summary Enzootic bovine leukosis is a contagious disease induced by bovine leukemia virus (BLV). It is a chronic disease that develops over a long period and can be schematically divided into three phases: (1) from birth to infection (some animals are already infected at birth), (2) from viral infection to tumorous transformation (a number of infected animals do not develop tumors before being slaughtered), and (3) from tumorous transformation to death. Bovine leukotic lymphocytes, kept in vitro in suitable medium, release detectable amounts of BLV. Short-term cultures of leukocytes from cows with persistent lymphocyotsis or long-term cultures of BLV-infected cells were used to study the morphogenesis of BLV, wherein one system was made of degenerating cells, the other made of healthy fast growing cells. Detection of anti-BLV antibodies includes immunodiffusion using p 24 , p 15 , immunofluorescence (IF), complement fixation (CF), and radioimmunoassay. All these methods are indirect detection methods. They detect BLV infection through the immune reaction of the host. The chapter presents the experiments performed to understand the transmission of BLV to the homologous host.

Use of monoclonal antibody in an ELISA test for the detection of antibodies to bovine leukaemia virus.

A variant of the ELISA technique, involving a monoclonal anti-gp51 antibody yields a highly sensitive method for the detection of bovine leukaemia virus (BLV) antibodies. The gp51 antigen-coated microtitre plates are obtained by incubation of plastic-adsorbed monoclonal antibodies with a non-purified mixture of BLV antigens. Sera to be tested are incubated in the wells of the gp51-coated plates and bound antibodies are revealed by an enzyme-linked antibovine immunoglobulin reagent. This test is as sensitive as liquid phase radioimmunoassay using the same gp51 antigen and thus appears as a highly sensitive, practical, rapid and cheap method for the detection of BLV antibodies.

In animals infected by bovine leukemia virus (BLV) antibodies to envelope glycoprotein gp51 are directed against the carbohydrate moiety.

Abstract The immunological reactivity of the surface glycoprotein gp51 of bovine leukemia virus was examined by radioimmunoassay with sera from infected animals and sera obtained from rabbits after injection of the purified antigen. The respective influence of the protein and carbohydrate portions of the glycoprotein on the antigenic reactivity was investigated by digestions with glycosidases and proteases. Digestion of gp51 with a mixture of glycosidases abolished the reactivity of the antigen with sera of infected cattle or sheep. In contrast, the reactivity of gp51 with monospecific rabbit antisera was only slightly modified by the glycosidase treatment. Digestion of the same antigen with proteinase K and Pronase completely eliminated its reactivity with monospecific rabbit antiserum; sera from infected cattle or sheep still precipitated 10 to 15% of the same 125 I-antigen in direct radioimmunoassay. These results strongly indicate that natural immunity against BLV gp51 depends upon an intact carbohydrate side chain. Following purification of the antigen, protein antigenic sites are uncovered and reacted against by the injected rabbit. Solid phase radioimmunoassay furthermore showed that the carbohydrate antigenic site is probably unique as opposed to the probably multiple protein antigenic determinants. Antibody to gp51 prepared in the rabbit by injection of intact BLV-infected bovine lymphocytes reacted, in all tests performed, in much the same way as natural antibody found in BLV-infected cattle or sheep.

Biologically active epitopes of Bovine Leukemia Virus glycoprotein gp51 : their dependence on protein glycosylation and genetic variability.

A panel of monoclonal antibodies to the bovine leukemia virus envelope glycoprotein (BLV gp51) has previously demonstrated the association of the biological activities of the virus (infectivity, syncytia induction) with three out of eight epitopes of gp51. In BLV-infected cells, the unglycosylated homolog of the precursor to the BLV envelope glycoproteins (gPr72env) is a 47,000-MW polypeptide. Immunoprecipitation studies with monoclonal antibodies show that the neutralizing antibody-inducing sites, although present in gPr72env, are not conserved in the 47,000-MW unglycosylated homolog. Finally, it is demonstrated that the neutralizing antibody-inducing sites of gp51 are subject to antigenic variation among BLV isolates of the same or different geographical origins.

Genomic integration of bovine leukemia provirus and lack of viral RNA expression in the target cells of cattle with different responses to BLV infection.

Enzootic bovine leukosis (EBL) is a contagious lymphoproliferative disease whose etiological agent is a retrovirus, the bovine leukemia virus (BLV). EBL is a complex disease. Soon after infection a strong humoral antibody response develops and persists for the animal’s entire life. Such BLV-infected cattle can remain asymptomatic virus carriers for many years. They can also at a given time develop persistent lymphocytosis (PL) characterized by a permanent large number of peripheral lymphocytes. A variable but always significant percentage of PL animals develop lymphoid tumors, the terminal tumor phase of EBL. The remnant tumor cases develop suddenly in BLV carriers without any previous hematologic disorder. In general, the fate of BLV-infected animals is variable and depends upon several factors, including age, genetic make-up, environmental factors, and immunologic surveillance (see Burny et al. 1980 for a review).

Use of two monoclonal antibodies in an ELISA test for the detection of antibodies to bovine leukaemia virus envelope protein gp51.

A competition ELISA technique involving two monoclonal anti-gp51 antibodies has been developed for the detection of bovine leukaemia virus (BLV) antibodies. Precoated gp51 antigen-microtitre plates were obtained by incubation of plastic adsorbed monoclonal antibody with a non-purified BLV preparation. Samples to be tested were incubated in the wells of the gp51-coated plates; the presence of anti-gp51 antibodies was indicated by competition for antigen binding with an enzyme linked monoclonal antibody directed to an important epitope on gp51. This test is as sensitive as a routinely used indirect ELISA test; it is highly specific, reliable and easy to perform.

Antigenic variants of bovine leukemia virus (BLV) are defined by amino acid substitutions in the NH2 part of the envelope glycoprotein gp51

Previous studies with monoclonal antibodies of the antigenic structure of bovine leukemia virus (BLV) envelope glycoprotein (gp51) have identified three epitopes (F, G, H) directly involved in the infectivity of BLV, F, G, and H lost their reactivity with the respective monoclonal antibodies after treatment with a reducing agent, indicating that these epitopes were conformational. Sequence comparisons between BLV mutants and differential reactivities of urokinase or proteinase K gp51 fragments with monoclonal antibodies indicated that the NH2 moiety of the env protein harbored the three architectural determinants F, G, and H. ELISA tests demonstrated that anti-F, -G, and -H monoclonal antibodies were maximally reactive toward intact virions whereas they showed much poorer affinities for their respective epitopes when presented on a purified protein. Accordingly, an efficient vaccine against BLV infection will include at least the identified gp51 region presented in its native architectural configuration.

In vivo transfection of bovine leukemia provirus into sheep

Bovine leukemia virus is horizontally transmitted mainly through infected cells by direct blood transfer. In this report, a cloned bovine leukemia virus (BLV) provirus was examined for its infectivity by direct inoculation into sheep. One hundred micrograms of plasmid DNA containing a complete provirus was mixed with a cationic liposome solution and injected intradermally into five sheep at three different locations. Seroconversion occurred 1 to 2 months after injection as demonstrated by immunodiffusion, indirect ELISA (for the gp51 envelope protein), and blocking ELISA (for gp51 and the major capsid protein, p24). These results demonstrate that BLV infection can be efficiently initiated by direct transfection into sheep. This approach should thus facilitate investigation of the involvement of BLV genetic determinants in the induction of leukemia in ruminants.

Experimental transmission of enzootic bovine leukosis to cattle, sheep and goats: Infectious doses of blood and incubation period of the disease

A group of 49 BLV-free recipient animals (24 cattle, 15 sheep and 10 goats) were inoculated intradermally with serial dilutions of blood collected on two BLV-positive donor cows. One donor had a high lymphocytosis and high antibody titers to gpBLV antigens; these two parameters were low for the second donor. The number of lymphocytes which induced BLV infection in recipient animals varied widely with the donor. The high infectivity of a donor seemed to be correlated with high lymphocytosis and high antibody titers to gpBLV antigens. Identification and removal of infectious animals would reduce or stop the spread of the infection in a herd, and could be used in the strategy to eradicate the disease. A given inoculum can be infectious in sheep and, at the same time, harmless in cattle. The incubation periods, apparently shorter in sheep, were generally in the range from 2 to 5 weeks for the three species, and exceptionally above.

Recombinant vaccinia virus expression of the bovine leukaemia virus envelope gene and protection of immunized sheep against infection

The bovine leukaemia virus (BLV) envelope gene encoding extracellular glycoprotein gp51 and transmembrane glycoprotein gp30 was cloned into the HA locus of vaccinia virus (Copenhagen strain), downstream of the vaccinia virus early-late promoter, H6, or a triple promoter element consisting of the promoter for the vaccinia virus H6 gene, the promoter for the cowpox virus A-type inclusion (ATI) gene and the promoter for the vaccinia virus HA gene. Inoculation of rabbits or sheep with the recombinant vaccinia virus coding for gp51 and gp30 or an uncleaved env precursor induced neutralizing antibodies to BLV. These antibodies competed with monoclonal antibodies directed against gp51 epitopes F, G, and H previously shown to be of crucial importance for BLV infection. Seven out of eight sheep vaccinated with the vaccinia recombinants resisted a drastic challenge (1.5 x 10(3) sheep infectious doses) with BLV-infected lymphocytes. These results show that vaccination with BLV env vaccinia recombinants protects sheep against infection with extremely high doses of BLV-infected heterologous lymphocytes.