An essential role for γ-herpesvirus latency-associated nuclear antigen homolog in an acute lymphoproliferative disease of cattle.

Wildebeests carry asymptomatically alcelaphine herpesvirus 1 (AlHV-1), a γ-herpesvirus inducing malignant catarrhal fever (MCF) to several ruminant species (including cattle). This acute and lethal lymphoproliferative disease occurs after a prolonged asymptomatic incubation period after transmission. Our recent findings with the rabbit model indicated that AlHV-1 infection is not productive during MCF. Here, we investigated whether latency establishment could explain this apparent absence of productive infection and sought to determine its role in MCF pathogenesis. First, whole-genome cellular and viral gene expression analyses were performed in lymph nodes of MCF-developing calves. Whereas a severe disruption in cellular genes was observed, only 10% of the entire AlHV-1 genome was expressed, contrasting with the 45% observed during productive infection in vitro. In vivo, the expressed viral genes included the latency-associated nuclear antigen homolog ORF73 but none of the regions known to be essential for productive infection. Next, genomic conformation analyses revealed that AlHV-1 was essentially episomal, further suggesting that MCF might be the consequence of a latent infection rather than abortive lytic infection. This hypothesis was further supported by the high frequencies of infected CD8(+) T cells during MCF using immunodetection of ORF73 protein and single-cell RT-PCR approaches. Finally, the role of latency-associated ORF73 was addressed. A lack of ORF73 did not impair initial virus replication in vivo, but it rendered AlHV-1 unable to induce MCF and persist in vivo and conferred protection against a lethal challenge with a WT virus. Together, these findings suggest that a latent infection is essential for MCF induction.

Production and utilization of interleukin-15 in malignant catarrhal fever.

Malignant catarrhal fever (MCF) is an often fatal lymphoproliferative disease of ungulates caused by either alcelaphine herpesvirus-1 (AlHV-1) or ovine herpesvirus-2 (OvHV-2). The pathogenesis of MCF is poorly understood, but appears to involve an auto-destructive pathology whereby cytotoxic lymphocytes destroy areas of a variety of tissues. The cytokine interleukin-15 (IL-15) is involved in the development and maintenance of cytotoxic lymphocytes and may therefore have a role in the pathogenesis of MCF. Virus-infected large granular lymphocytes (LGLs) were obtained from the tissues of rabbits infected with AlHV-1 or OvHV-2. These cells exhibited a similar proliferative response to IL-15 and to IL-2 in culture, but their content of the activated cytotoxic enzyme (BLT-esterase) was maintained at higher levels in the presence of IL-15 compared with IL-2. The LGLs did not express IL-15 mRNA or produce IL-15 protein. By contrast, there was abundant expression of IL-15 mRNA and protein in affected tissues. IL-15 production was associated with necrotic lesions of the mesenteric lymph node and appendix of OvHV-2-infected rabbits, but was not found in the same tissues of rabbits infected with AlHV-1 in which there were no necrotic lesions. The cellular source of the IL-15 was predominantly lymphoid cells that did not express B cell or monocyte-macrophage markers. Only a few IL-15+ cells (<10%) co-localized with pan-T cells or CD8+ T cells. The abundance of IL-15 in tissue with lesions of MCF suggests that this cytokine may have a role in the pathogenesis of MCF.

The A5 gene of alcelaphine herpesvirus 1 encodes a constitutively active G-protein-coupled receptor that is non-essential for the induction of malignant catarrhal fever in rabbits.

Many gammaherpesviruses encode G-protein-coupled receptors (GPCRs). Several in vivo studies have revealed that gammaherpesvirus GPCRs are important for viral replication and for virus-induced pathogenesis. The gammaherpesvirus alcelaphine herpesvirus 1 (AlHV-1) is carried asymptomatically by wildebeest, but causes malignant catarrhal fever (MCF) following cross-species transmission to a variety of susceptible species. The A5 ORF of the AlHV-1 genome encodes a putative GPCR. In the present study, we investigated whether A5 encodes a functional GPCR and addressed its role in viral replication and in the pathogenesis of MCF. In silico analysis supported the hypothesis that A5 could encode a functional GPCR as its expression product contained several hallmark features of GPCRs. Expression of A5 as tagged proteins in various cell lines revealed that A5 localizes in cell membranes, including the plasma membrane. Using [35S]GTPgammaS and reporter gene assays, we found that A5 is able to constitutively couple to alpha i-type G-proteins in transfected cells, and that this interaction is able to inhibit forskolin-triggered cAMP response element-binding protein (CREB) activation. Finally, using an AlHV-1 BAC clone, we produced a strain deleted for A5 and a revertant strain. Interestingly, the strain deleted for A5 replicated comparably to the wild-type parental strain and induced MCF in rabbits that was indistinguishable from that of the parental strain. The present study is the first to investigate the role of an individual gene of AlHV-1 in MCF pathogenesis.

Immunohistochemical study of experimental malignant catarrhal fever in rabbits.

Malignant catarrhal fever (MCF) is an often-fatal lymphoproliferative disease of a variety of ungulates that occurs worldwide. It is caused by either of the highly related but distinct gammaherpesviruses alcelaphine herpesvirus-1 (AlHV-1, wildebeest reservoir) or ovine herpesvirus-2 (OvHV-2, sheep reservoir). MCF in rabbits is an excellent model as it closely resembles the disease in susceptible ungulates that include cattle, deer and bison. In this study, newly available and previously characterized monoclonal antibodies specific for rabbit leucocyte differentiation molecules were used to perform a detailed immunohistochemical examination of both AlHV-1 MCF and OvHV-2 MCF in rabbits. Differences in the MCF caused by the two viruses included: less tissue necrosis and more lymphoid cell accumulations in AlHV-1 MCF compared with OvHV-2 MCF, and in particular marked tissue necrosis in the mesenteric lymph node, appendix and liver of OvHV-2-infected animals when compared with either other tissues in OvHV-2 MCF or AlHV-1 MCF lesions in any tissue. In both AlHV-1 MCF and OvHV-2 MCF, lymphoid cell accumulations in lymphoid and non-lymphoid tissues consisted mainly of T-cells with a corresponding absence of B-cells. CD8(+) T-cells accounted for a proportion of these in the non-lymphoid tissues, but there was evidence for the accumulation of an unidentified T-cell subset/subsets as well. This study extends our understanding of the mechanisms of immuno-pathogenesis of MCF.