Bovine leukemia virus encoded blv-miR-b4-3p microRNA is associated with reduced expression of anti-oncogenic gene in vivo.

Bovine leukemia virus (BLV) is a retrovirus that causes malignant B-cell lymphoma in up to ten-percent of infected cattle. To date, the mechanisms of BLV linked to malignant transformation remain elusive. Although BLV-encoded miRNAs have been associated with the regulation of different genes involved in oncogenic pathways, this association has not been evaluated in cattle naturally infected with BLV. The objective of this study was to determine the relative expression of BLV-encoded miRNA blv-miR-b4-3p, the host analogous miRNA bo-miR-29a and a couple of potential target mRNAs (HBP-1 and PXDN, with anti-tumorigenic function in B-cells), in cattle naturally infected with BLV compared to uninfected animals (control group). We observed that PXDN was significantly downregulated in BLV-infected cattle (P = 0.03). Considering the similar expression of endogenous bo-miR-29a in both animal groups, the downregulation of PXDN in BLV-naturally infected cattle could be linked to blv-miR-b4-3p expression in these animals. Knowing that PXDN is involved in anti-tumoral pathways in B-cells, the results presented here suggest that blv-miR-b4-3p might be involved in BLV tumorigenesis during natural infection with BLV in cattle.

A safe and effective vaccine against bovine leukemia virus.

Previous attempts to develop a vaccine against bovine leukemia virus (BLV) have not been successful because of inadequate or short-lived stimulation of all immunity components. In this study, we designed an approach based on an attenuated BLV provirus by deleting genes dispensable for infectivity but required for efficient replication. The ability of the vaccine to protect from natural BLV infection was investigated in the context of dairy productive conditions in an endemic region. The attenuated vaccine was tested in a farm in which the prevalence rose from 16.7% in young cattle at the beginning of the study to more than 90% in adult individuals. Sterilizing immunity was obtained in 28 out of 29 vaccinated heifers over a period of 48 months, demonstrating the effectiveness of the vaccine. As indicated by the antiviral antibody titers, the humoral response was slightly reduced compared to wild-type infection. After initial post-vaccination bursts, the proviral loads of the attenuated vaccine remained most frequently undetectable. During the first dairy cycle, proviral DNA was not detected by nested-PCR in milk samples from vaccinated cows. During the second dairy cycle, provirus was sporadically detected in milk of two vaccinated cows. Forty-two calves born from vaccinated cows were negative for proviral DNA but had antiviral antibodies in their peripheral blood. The attenuated strain was not transmitted to sentinels, further supporting the safety of the vaccine. Altogether, these data thus demonstrate that the vaccine against BLV is safe and effective in herd conditions characterized by a very high incidence. This cost-effective approach will thus decrease the prevalence of BLV without modification of production practices. After facing a series of challenges pertaining to effectiveness and biosafety, the vaccine is now available for further large-scale delivery. The different challenges and hurdles that were bypassed may be informative for the development of a vaccine against HTLV-1.

Development of a droplet digital PCR assay for quantification of the proviral load of bovine leukemia virus.

Droplet digital PCR (ddPCR) is a highly sensitive tool developed for the detection and quantification of short-sequence variants-a tool that offers unparalleled precision enabling measurement of smaller-fold changes. We describe here the use of ddPCR for the detection of Bovine leukemia virus (BLV) DNA provirus. Serum samples and whole blood from experimentally infected sheep and naturally infected cattle were analyzed through ddPCR to detect the BLV gp51 gene, and then compared with serologic and molecular tests. The ddPCR assay was significantly more accurate and sensitive than AGID, ELISA, nested PCR, and quantitative PCR. The limit of detection of ddPCR was 3.3 copies/µL, detecting positive experimentally infected sheep beginning at 6 d post-infection. The ddPCR methodology offers a promising tool for evaluating the BLV proviral load, particularly for the detection of low viral loads.

BLV: lessons on vaccine development.

Vaccination against retroviruses is a challenge because of their ability to stably integrate into the host genome, undergo long-term latency in a proportion of infected cells and thereby escape immune response. Since clearance of the virus is almost impossible once infection is established, the primary goal is to achieve sterilizing immunity. Besides efficacy, safety is the major issue since vaccination has been associated with increased infection or reversion to pathogenicity. In this review, we discuss the different issues that we faced during the development of an efficient vaccine against bovine leukemia virus (BLV). We summarize the historical failures of inactivated vaccines, the efficacy and safety of a live-attenuated vaccine and the economical constraints of further industrial development.

Spontaneous virus reactivation in cattle chronically infected with bovine leukemia virus.

BACKGROUND: The absence of virus expression during the chronic stage of bovine leukemia virus (BLV) infection and its reactivation upon ex vivo culture has become a long-lived Dogma. During the chronic stage of BLV infection the immune response limits viral replication and the mitotic division of latently infected cells, carrying BLV provirus, allows viral expansion and disease progression towards a lymphoproliferative disorder. Several stressor factors have been associated with animal production and handling. As natural mediator of stress, glucocorticoids are strong immunosuppressive agents; moreover, they can bind long-terminal repeat region of retroviruses and induce viral expression. In the present study, we present a case report describing the spontaneous reactivation of BLV infection in naturally infected cattle. CASE PRESENTATION: In order to investigate if virus reactivation occurred in vivo during the course of BLV infection, we followed up for 328 days one Holstein cow (> 3 years) chronically infected with BLV which presented high-proviral loads. This animal was neither lactating nor pregnant. Furthermore, we investigated if a stressor stimulus, in this case the administration of a synthetic glucocorticoid (dexamethasone), could impact the course of BLV infection in three additional cattle. For the first time, we observed a high level of BLV transcripts in a total of four cattle chronically infected with BLV. The detection of viral transcripts corresponding to pol gene strongly suggests virus reactivation in these animals. Interestingly, this simultaneous virus reactivation was unrelated to dexamethasone treatment. CONCLUSIONS: We reported for the first time spontaneous and high level of BLV transcriptional activation in cattle chronically infected with BLV. Although virus reactivation was unrelated to dexamethasone treatment, other stressor stimuli might have influenced this outcome. Future studies will be necessary to understand these observations, since the spontaneous virus reactivation presented here might have implications on BLV pathogenesis and transmission.

Evidence of reduced vertical transmission of Neospora caninum associated with higher IgG1 than IgG2 serum levels and presence of IFN-γ in non-aborting chronically infected cattle under natural condition.

Neospora caninum infection of cattle can be vertically transmitted, resulting in abortion or birth of infected calves. Vertical transmission occurs both in acutely or chronically infected cattle. There is little information on the immune response needed to prevent endogenous transplacental transmission, particularly from chronically infected cattle to their offspring in a natural environment. In this study, N. caninum seropositive pregnant cattle from three different farms with high avidity antibodies and low IgM titers were selected and their newborn colostrum-deprived calves were tested for anti-N. caninum antibodies. Based on these results, dams were grouped according to their congenital transmission status. The analysis of the immune profile of the chronically-infected pregnant cattle revealed that higher ratio between IgG1 and IgG2 anti-N. caninum serum titers and higher levels of systemic IFN-γ were associated with diminished vertical transmission rates, compared to dams with the opposite profile. Our results evidenced an association between the immune profile and vertical transmission in non-aborting chronically infected dams, and confirm that vertical transmission, even when not leading to abortion, is related to a defined immune profile. This is important information to accomplish successful vaccine development efforts.