Development of an indirect ELISA based on recombinant capsid protein to detect antibodies to bovine leukemia virus

Abstract Serological testing and culling infected animals are key management practices aiming eradication of bovine leukemia virus infection. Here, we report the development of an indirect ELISA based on BLV recombinant capsid protein (BLVp24r) to detect anti-BLV antibodies in cattle serum. The BLVp24r was expressed in Escherichia coli and purified by affinity chromatography, and then used to set up the ELISA parameters. The Polysorp ® plate coated with 50 ng of antigen/well and bovine serum diluted 1:100 gave the best results during standardization. Using sera from infected and non-infected cattle we set up the cutoff point at 0.320 (OD450 nm) with a sensitivity of 98.5% and specificity of 100.0%. Then, we tested 1.187 serum samples from dairy (736 samples) and beef cattle (451 samples) with unknown status to BLV. We found that 31.1% (229/736) and 9.5% (43/451) of samples amongst dairy and beef cattle, respectively, had IgGs to BLV. The rate of agreement with a commercial competitive ELISA was 84.3% with a κ value of 0.68. Thus, our BLVp24r iELISA is suitable to detect BLV infected animals and should be a useful tool to control BLV infection in cattle.

A novel association of BoLA DRB3 alleles in BLV infected cattle with different proviral loads / Uma nova associação de alelos de BoLA DRB3 em bovinos infectados com BLV com diferentes cargas provirais

Bovine leukemia virus (BLV) is associated with the most common neoplastic disease of cattle. BLV has a silent dissemination in the herd due to infected cell exchange, thus the concentration of BLV-infected cells in blood should play a major role in the success of viral transmission. Genes from Bovine leukocyte antigen (BoLA), the MHC system of cattle, are associated with genetic resistance and susceptibility to a wide range of diseases, and also with production traits. Some BoLA DRB3.2 allele polymorphisms in Holstein cattle have been associated with resistance or susceptibility to BLV-disease development, or with proviral load (PVL). This investigation studied 107 BLV-infected Argentinean Holstein dairy cows, all of them belonging to one herd. PVL was analysed by qPCR and animals were classified as high proviral load (HPVL, N = 88) and low proviral load (LPVL, N = 19), and BoLA DRB3.2 alleles were genotyped. Alleles BoLA DRB3.2*1501 and *1201 were significantly associated with HPVL (p = 0.0230 and p = 0.0111 respectively), while allele BoLA DRB3.2*0201 was significantly associated with LPVL (p = 0.0030). The present study aims at contributing to the knowledge of the association between BoLA polymorphism and development of a BLV infection profile. Genes that best explain the PVL in this population resulted BoLA DRB3.2*0201 (as a protection factor) and *1501 (as a risk factor). Allelic differences may play an important role in the development of effective immune responses. A better understanding of how BoLA polymorphism contributes to these responses and the establishment of a BLV status is desirable to schedule and evaluate control measures.(AU)

O vírus da leucemia bovina (BLV) está associado à doença neoplásica mais comum do gado bovino. O BLV tem uma disseminação silenciosa no rebanho devido à troca de células infectadas, assim, a concentração de células BLV infectadas no sangue deve desempenhar um papel importante no sucesso da transmissão viral. Os genes do antígeno leucocitário bovino (BoLA), sistema MHC do gado bovino, estão associados à resistência genética e à susceptibilidade a uma ampla gama de doenças, bem como às características da produção. Alguns polimorfismos de alelos de BoLA DRB3.2 em bovinos Holstein têm sido associados à resistência ou susceptibilidade ao desenvolvimento da doença BLV, ou com carga proviral (PVL). Esta investigação avaliou 107 vacas leiteiras da raça Holstein argentina infectadas com BLV e pertencentes a um único rebanho. A PVL foi analisada por qPCR, os animais foram classificados em alta carga proviral (HPVL, N = 88) e baixa carga proviral (LPVL, N = 19), e os alelos BoLA DRB3.2 foram genotipados. Os alelos BoLA DRB3.2*1501 e *1201 estavam significativamente relacionados à HPVL (p = 0,0230 e p = 0,0111, respectivamente), enquanto o alelo BoLA DRB3.2*0201, à LPVL (p = 0,0030). O objetivo deste estudo é contribuir para o conhecimento da associação entre o polimorfismo de BoLA e o desenvolvimento de infecção por BLV. Os genes que melhor explicam a PVL na população analisada resultaram em BoLA DRB3.2*0201 (como fator de proteção) e *1501 (como fator de risco). As diferenças alélicas podem desempenhar um papel importante no desenvolvimento de respostas imunitárias eficazes. Uma melhor compreensão de como o polimorfismo BoLA contribui para estas respostas e o estabelecimento de um estado BLV é desejável para agendar e avaliar as medidas de controle.(AU)

Agar gel immunodiffusion analysis using baculovirus-expressed recombinant bovine leukemia virus envelope glycoprotein (gp51/gp30T-)

Bovine leukemia virus (BLV) envelope glycoprotein (gp51/gp30T-), consisting of BLV gp51 and BLV gp30 that lacked its C-terminal transmembrane domain, was expressed in insect cells under the control of the baculovirus polyhedron promoter. Recombinant BLV gp51/gp30T- secreted from insect cells was determined by immunofluorescence, enzyme-linked immunosorbent and western blot assays using a BLV-specific monoclonal antibody and BLV-positive bovine antibodies. An agar gel immunodiffusion (AGID) test using gp51/gp30T- as the antigen for the detection of BLV antibodies in serum was developed and compared to traditional AGID, which uses wild type BLV antigen derived from fetal lamb kidney cells. AGID with the recombinant BLV gp51/gp30T- was relatively more sensitive than traditional AGID. When the two methods were tested with bovine sera from the field, the recombinant BLV gp51/gp30T- and traditional antigen had a relative sensitivity of 69.8% and 67.4%, respectively, and a relative specificity of 93.3% and 92.3%. These results indicated that the recombinant BLV gp51/gp30T- is an effective alternative antigen for the diagnosis of BLV infection in cattle.

Simultaneous presence of bovine papillomavirus and bovine leukemia virus in different bovine tissues: in situ hybridization and cytogenetic analysis

Bovine papillomavirus (BPV) DNA sequences were detected in different tissues, in addition to epithelium. Cytogenetic abnormalities were observed in blood lymphocytes. The presence of more than one virus in a single tissue is a difficult aspect to evaluate, especially when the DNA sequences are detected in tissues that are not specifically targeted by the virus. BPV and bovine leukemia virus (BLV) are clastogenic, causing chromosome aberrations in peripheral blood lymphocytes. In the present study, we investigated the simultaneous presence of DNA sequences of both viruses and the possibility of vertical transmission and compared the types of chromosome aberrations related to viral action. BPV 1, 2, and 4 DNA sequences were found in three females of the herd and in their offspring. BLV DNA sequences were not detected in their progeny. A newborn calf that was negative for BLV infection showed specific chromosome rearrangements possibly related to the effect of infection with BPV.

Investigation of the Bovine Leukemia Virus Proviral DNA in Human Leukemias and Lung cancers in Korea

The bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis. This study investigated the presence of the BLV in leukemia (179 acute lymphoblastic leukemia, 292 acute myeloid leukemia and 46 chronic myelogenous leukemia cases) and 162 lung cancer patients (139 adenocarcinoma, 23 squamous cell carcinoma) to determine if the BLV is a causative organism of leukemia and lung cancer in Koreans. A BLV infection was confirmed in human cells by PCR using a BLV-8 primer combination. All 517 cases of human leukemia and 162 lung cancer were negative for a PCR of the BLV proviral DNA. In conclusion, although meat has been imported from BLV endemic areas, the BLV infection does not appear to be the cause of human leukemia or lung cancer in Koreans. These results can be used as a control for further studies on the BLV in Koreans.

Detection of px gene product of bovine leukemia virus in infected cells.

Bovine leukemia virus (BLV), like its closest relatives human T-cell leukemia virus-I and II, contain a 'px' gene, between the 'env' gene and the 3' long terminal repeat in its genome. A monoclonal antibody prepared against a synthetic oligopeptide whose sequence was deduced from highly conserved region of 'px' gene of BLV, was used to detect the presence of 'px' gene product in chronically BLV infected synchronised cells. By immunoperoxidase staining the 'px' gene product was detected maximum after 6-9 hr after synchronization in the nucleus of the cells which demonstrated the close interaction of it with viral DNA which is integrated with host cell genome.

Binding of phytohemagglutinin to bovine B lymphocytes and its role in stimulation of expression of bovine leukemia virus genome.

Phytohemagglutinin (PHA) is known to increase the synthesis of bovine leukemia virus (BLV) particles and viral antigens in short-term culture of BLV-infected neoplastic and non-neoplastic lymphocytes. This stimulation of BLV expression has been shown to be due to enhanced transcription of the viral genome by a PHA-induced protein. We have investigated the binding of 125I-labelled PHA to BLV-infected bovine B lymphocytes and subsequent events that may lead to the stimulation of BLV-p25 synthesis. We found that PHA binding to the infected cells were rapid, but only a small fraction of the bound PHA is translocated to nucleus. However, bound PHA dissociated rapidly from the cell membrane, but not from the nucleus when PHA is removed from the culture medium. Furthermore, continuous presence of PHA was not essential for optimal stimulatory activity, instead a minimum incubation with PHA for 6 hr. followed by culturing the infected cells in its absence, was sufficient to exhibit maximal stimulatory activity. Our results raise the possibility that interaction of PHA with the plasma membrane probably triggers synthesis of a protein which in turn enhances the transcription of BLV genome in vitro.

Leucosis bovina / Bovine leucosis

The enzootic form of bovine lymphosarcoma is the most common malignancy of cattle, especially dairy cattle. Its etiological agent, the bovine leukemia virus (BLV) is evolutionarily related to human T-cell leukemia viruses types I and II (HTLV and HTLV II). These three retroviruses share common structural and biological features that distingush them from other members of the retroviral family. BLV is widespread in the cattle population of most countries. However, clinical disease occurs much less frecuently and direct economic losses from morbidity and mortality are significant for individual cattle owners but not excessively burdensome to the cattle and meat packing industries. The primary economic loss to the cattle industry from bovine leukosis is tha actual and potential loss of export markets for cattle and semen. BLV can infect human and simian cells "in vitro", there is serological evidence that BLV infect chimpanzes and in the literature can be found circunstantial evidence for an association of bovine and human leukemia. There is hight incidence of BLV infectión in dairy cattle and the virus is excreted in milk. At present, no evidence had been found that BLV or infected cattle are human health hazards, however, any conclusion would be premature. To clarify this issue investigations applying the most sensitive methods will be necessary. Several sensitive and specific methods for the detection og BLV and BLV infected cattle have been developed. These include infectivity assays, serological tests, and molecular hydridization techniques. Since under natural conditions BLV is predominantly transmitted by contact and prenatural conditions BLV is predominantly transmitted by contact and prenatal infection occurs in less than 20% of calves born to infected dams, control and/or erradication programs wuld be feasibly