- Volume 95, Issue 8, 2014

Recently, we immunized different mammalian species (goats, mice, rats, rabbits, guinea pigs and hamsters) with the recombinant ectodomain of the transmembrane envelope (TM) protein p15E of porcine endogenous retrovirus (PERV). In all cases, neutralizing immune sera were induced, which recognized epitopes in the fusion peptide proximal region and the membrane proximal external region of p15E. In order to analyse whether pigs are also able to produce such antibodies, and whether such antibodies can be used to study the involvement of the TM protein in placental development (as was shown for endogenous retroviruses of other species), German landrace pigs were immunized with PERV p15E. No binding and neutralizing antibodies were produced as shown in three Western blot analyses and in a neutralization assay, indicating that pigs are tolerant to their endogenous retroviruses, at least for the ectodomain of the TM protein.

Bovine leukemia virus (BLV) induces abnormal B-cell proliferation and B-cell lymphoma in cattle, where the BLV provirus is integrated into the host genome. BLV-infected B-cells rarely express viral proteins in vivo, but short-term cultivation augments BLV expression in some, but not all, BLV-infected B-cells. This observation suggests that two subsets, i.e. BLV-silencing cells and BLV-expressing cells, are present among BLV-infected B-cells, although the mechanisms of viral expression have not been determined. In this study, we examined B-cell markers and viral antigen expression in B-cells from BLV-infected cattle to identify markers that may discriminate BLV-expressing cells from BLV-silencing cells. The proportions of IgMhigh B-cells were increased in blood lymphocytes from BLV-infected cattle. IgMhigh B-cells mainly expressed BLV antigens, whereas IgMlow B-cells did not, although the provirus load was equivalent in both subsets. Several parameters were investigated in these two subsets to characterize their cellular behaviour. Real-time PCR and microarray analyses detected higher expression levels of some proto-oncogenes (e.g. Maf, Jun and Fos) in IgMlow B-cells than those in IgMhigh B-cells. Moreover, lymphoma cells obtained from the lymph nodes of 14 BLV-infected cattle contained IgMlow or IgM− B-cells but no IgMhigh B-cells. To our knowledge, this is the first study to demonstrate that IgMhigh B-cells mainly comprise BLV-expressing cells, whereas IgMlow B-cells comprise a high proportion of BLV-silencing B-cells in BLV-infected cattle.

Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of the nasal mucosa of sheep and goats and is associated with enzootic nasal tumour virus (ENTV). As ENA is a common disease in North America and there are no vaccines against ENTV-1, diagnostic tests that can identify infected animals and assist with eradication and disease surveillance efforts are greatly needed. In this study, we endeavoured to develop a novel, non-invasive diagnostic tool that could be used not only to validate clinical signs of ENA but also to detect ENTV-1 infection prior to the onset of disease signs (i.e. pre-clinical diagnosis). Cytology, serology and reverse transcription (RT)-PCR-based diagnostic methods were investigated. Although the cytology-based assay was able to detect ENTV-1 infection in some animals, it had poor sensitivity and specificity and thus was not developed further as an ante-mortem diagnostic method. Three different assays, including ELISA, Western blotting and virus neutralization, were developed to detect the presence of ENTV-1-specific antibodies in sheep serum. Whilst a surprisingly large number of sheep mounted an antibody-mediated immune response against ENTV-1, and in some cases neutralizing, correlation with disease status was poor. In contrast, RT-PCR on RNA extracted from nasal swabs reliably detected exogenous ENTV-1 sequences, did not amplify endogenous ovine betaretroviral sequences, demonstrated high concordance with immunohistochemical staining for ENTV-1 envelope protein, and had perfect sensitivity and specificity. This report describes a practical and highly specific RT-PCR technique for the detection of clinical and pre-clinical ENA that may prove beneficial in future control or eradication programmes.