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

in Proceedings of the National Academy of Sciences of the United States of America (2013)

Wildebeests carry asymptomatically alcelaphine herpesvirus 1 (AlHV-1), a gamma-herpesvirus inducing malignant catarrhal fever (MCF) to several ruminant species (including cattle). This acute and lethal ... [more ▼]

Wildebeests carry asymptomatically alcelaphine herpesvirus 1 (AlHV-1), a gamma-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. [less ▲]

Investigation on the Role of the Viral Semaphorin encoded by the A3 Gene of Alcelaphine Herpesvirus 1 in the Induction of Malignant Catarrhal Fever

Poster (2011, July)

Alcelaphine herpesvirus 1 (AlHV-1) is a gammaherpesvirus carried by wildebeest asymptomatically. AlHV-1 is however responsible for the development of malignant catarrhal fever (MCF) when cross-species ... [more ▼]

Alcelaphine herpesvirus 1 (AlHV-1) is a gammaherpesvirus carried by wildebeest asymptomatically. AlHV-1 is however responsible for the development of malignant catarrhal fever (MCF) when cross-species transmitted to a variety of ruminant susceptible species. Wildebeest-derived (WD)-MCF is a fatal lymphoproliferative and degenerative disease of ruminants. Experimentally, WD-MCF can be reproduced in rabbits. A3 ORF encodes a putative semaphorin homolog protein, named AlHV-sema. Semaphorins are secreted and membrane-associated proteins characterized by a conserved ‘Sema’ domain. Initially identified as guidance factors assisting axons pathfinding during neural development, semaphorins have been shown over the last decade to have significant functions in various processes of immunoregulation. Phylogenetic analyses revealed that AlHV-sema and mammals Sema7A have a common ancestor and that AlHV-Sema has evolved independently of other viral semaphorins. Further bioinformatics analyses demonstrated that AlHV-Sema and cellular Sema7A share a highly similar tridimensional structure. In order to investigate the role of AlHV-Sema in WD-MCF induction, we used the AlHV-1 BAC clone and produced a strain deleted for A3 and a revertant strain. The strain deleted for A3 replicated comparably to the wild-type parental strain in vitro. In vivo, rabbits infected with the strain deleted for A3 developed WD-MCF with a small but significant delay compared to those infected with the parental and revertant strains. Deletion of A3 did not affect the increase of viral genomic charge over time in peripheral blood and in lymph nodes at time of death and the major histopathological lesions were present in all groups. Though infection with wild-type and revertant strains resulted in the inversion of CD8 over CD4 ratio and increased IFN- production in lymphoid tissues at time of death, both parameters were significantly reduced after infection with the A3 deleted strain. Together, these results suggest that AlHV-Sema play a role in the host response to AlHV-1 infection. [less ▲]

Ex vivo bioluminescent detection of Alcelaphine herpesvirus 1 infection during malignant catarrhal fever.

in Journal of Virology (2011), 85(14), 6941-54

Alcelaphine herpesvirus 1 (AlHV-1), carried by wildebeest asymptomatically, causes malignant catarrhal fever (WD-MCF) when cross-species transmitted to a variety of susceptible species of the Artiodactyla ... [more ▼]

Alcelaphine herpesvirus 1 (AlHV-1), carried by wildebeest asymptomatically, causes malignant catarrhal fever (WD-MCF) when cross-species transmitted to a variety of susceptible species of the Artiodactyla order. Experimentally, WD-MCF can be reproduced in rabbits. WD-MCF is described as a combination of lymphoproliferation and degenerative lesions in virtually all organs and caused by unknown mechanisms. Recently, we demonstrated that WD-MCF is associated with the proliferation of CD8(+) cells supporting a latent type of infection in lymphoid tissues. Here, we investigated the macroscopic distribution of AlHV-1 infection using ex vivo bioluminescence imaging in rabbit to determine whether it correlates with the distribution of lesions in lymphoid and non-lymphoid organs. To reach that goal, a recombinant AlHV-1 strain was produced by insertion of a luciferase expression cassette (luc) in an intergenic region. In vitro, the reconstituted AlHV-1 luc(+) strain replicated comparably to the parental strain and luciferase activity was detected by bioluminescence imaging. In vivo, rabbits infected with the AlHV-1 luc(+) strain developed WD-MCF comparably to the parental wild-type strain with hyperthermia and increase of both CD8(+) T cells frequencies and viral genomic charge over time in peripheral blood mononuclear cells and in lymph nodes at time of euthanasia. Bioluminescent imaging revealed that AlHV-1 infection could be detected ex vivo in lymphoid organs but also in lung, liver and kidney during WD-MCF, demonstrating that AlHV-1 infection is prevalent in tissue lesions. Finally, we show that the infiltrating mononuclear leukocytes in non-lymphoid organs are mainly CD8(+) T cells and that latency is predominant during WD-MCF. [less ▲]

Characterization of ovine herpesvirus 2-induced malignant catarrhal fever in rabbits

in Veterinary Microbiology (2011)

Malignant catarrhal fever (MCF) is a frequently fatal lymphoproliferative disease syndrome primarily of ruminant species, caused by gammaherpesviruses in the genus Macavirus. Ovine herpesvirus 2 (OvHV-2 ... [more ▼]

Malignant catarrhal fever (MCF) is a frequently fatal lymphoproliferative disease syndrome primarily of ruminant species, caused by gammaherpesviruses in the genus Macavirus. Ovine herpesvirus 2 (OvHV-2), carried by sheep, causes sheep-associated MCF worldwide, while Alcelaphine herpesvirus 1 (AlHV-1), carried by wildebeest, causes wildebeest-associated MCF, mainly in Africa. Diseases in rabbits can be induced by both viruses, which are clinically and pathologically similar; however, recent studies revealed different expression of viral genes associated with latency or lytic replication during clinical disease between the two viruses. In this study, we further characterized experimentally induced MCF in rabbits by nebulization with OvHV-2 from sheep nasal secretions to elucidate the course of viral replication, along with in vivo incorporation of 5-Bromo-2’-Deoxyuridine (BrdU), to evaluate lymphoproliferation. All six rabbits nebulized with OvHV-2 developed MCF between 24 and 29 days post infection. OvHV-2 DNA levels in peripheral blood leukocytes (PBL) remained undetectable during the incubation period and increased dramatically a few days before onset of clinical signs. During the clinical stage, we found that predominantly lytic gene expression was detected in PBL and tissues, and both T and B cells were proliferating. The data showed that the viral gene expression profile and lymphoproliferation in rabbits with OvHV-2 induced MCF were different from that in rabbits with AlHV-1 induced MCF, suggesting that OvHV-2 and AlHV-1 may play a different role in MCF pathogenesis. [less ▲]

IL-4Ralpha-responsive smooth muscle cells contribute to initiation of T(H)2 immunity and pulmonary pathology in Nippostrongylus brasiliensis infections.

in Mucosal immunology (2010), 4(1), 83-92

Nippostrongylus brasiliensis infections generate pulmonary pathologies that can be associated with strong T(H)2 polarization of the host's immune response. We present data demonstrating N. brasiliensis ... [more ▼]

Nippostrongylus brasiliensis infections generate pulmonary pathologies that can be associated with strong T(H)2 polarization of the host's immune response. We present data demonstrating N. brasiliensis-driven airway mucus production to be dependent on smooth muscle cell interleukin 4 receptor-alpha (IL-4Ralpha) responsiveness. At days 7 and 10 post infection (PI), significant airway mucus production was found in IL-4Ralpha(-/lox) control mice, whereas global knockout (IL-4Ralpha(-/-)) and smooth muscle-specific IL-4Ralpha-deficient mice (SM-MHC(Cre) IL-4Ralpha(-/lox)) showed reduced airway mucus responses. Furthermore, interleukin (IL)-13 and IL-5 cytokine production in SM-MHC(Cre) IL-4Ralpha(-/lox) mice was impaired along with a transient reduction in T-cell numbers in the lung. In vitro treatment of smooth muscle cells with secreted N. brasiliensis excretory-secretory antigen (NES) induced IL-6 production. Decreased protein kinase C (PKC)-dependent smooth muscle cell proliferation associated with cell cycle arrest was found in cells stimulated with NES. Together, these data demonstrate that both IL-4Ralpha and NES-driven responses by smooth muscle cells make important contributions in initiating T(H)2 responses against N. brasiliensis infections.Mucosal Immunology advance online publication 25 August 2010. doi:10.1038/mi.2010.46. [less ▲]

IL-4Ralpha-independent expression of mannose receptor and Ym1 by macrophages depends on their IL-10 responsiveness.

in PLoS Neglected Tropical Diseases (2010), 4(5), 689

IL-4Ralpha-dependent responses are essential for granuloma formation and host survival during acute schistosomiasis. Previously, we demonstrated that mice deficient for macrophage-specific IL-4Ralpha ... [more ▼]

IL-4Ralpha-dependent responses are essential for granuloma formation and host survival during acute schistosomiasis. Previously, we demonstrated that mice deficient for macrophage-specific IL-4Ralpha (LysM(cre)Il4ra(-/lox)) developed increased hepatotoxicity and gut inflammation; whereas inflammation was restricted to the liver of mice lacking T cell-specific IL-4Ralpha expression (iLck(cre)Il4ra(-/lox)). In the study presented here we further investigated their role in liver granulomatous inflammation. Frequencies and numbers of macrophage, lymphocyte or granulocyte populations, as well as Th1/Th2 cytokine responses were similar in Schistosoma mansoni-infected LysM(cre)Il4ra(-/lox) liver granulomas, when compared to Il4ra(-/lox) control mice. In contrast, a shift to Th1 responses with high IFN-gamma and low IL-4, IL-10 and IL-13 was observed in the severely disrupted granulomas of iLck(cre)Il4ra(-/lox) and Il4ra(-/-) mice. As expected, alternative macrophage activation was reduced in both LysM(cre)Il4ra(-/lox) and iLck(cre)Il4ra(-/lox) granulomas with low arginase 1 and heightened nitric oxide synthase RNA expression in granuloma macrophages of both mouse strains. Interestingly, a discrete subpopulation of SSC(high)CD11b+I-A/I-E(high)CD204+ macrophages retained expression of mannose receptor (MMR) and Ym1 in LysM(cre)Il4ra(-/lox) but not in iLck(cre)Il4ra(-/lox) granulomas. While aaMphi were in close proximity to the parasite eggs in Il4ra(-/lox) control mice, MMR+Ym1+ macrophages in LysM(cre)Il4ra(-/lox) mice were restricted to the periphery of the granuloma, indicating that they might have different functions. In vivo IL-10 neutralisation resulted in the disappearance of MMR+Ym1+ macrophages in LysM(cre)Il4ra(-/lox) mice. Together, these results show that IL-4Ralpha-responsive T cells are essential to drive alternative macrophage activation and to control granulomatous inflammation in the liver. The data further suggest that in the absence of macrophage-specific IL-4Ralpha signalling, IL-10 is able to drive mannose receptor- and Ym1-positive macrophages, associated with control of hepatic granulomatous inflammation. [less ▲]

Bovine herpesvirus 4 ORF73 is dispensable for viral growth in vitro but is essential for viral persistence in vivo.

in Journal of General Virology (The) (2010), 91(10), 2574-84

ORF73 orthologues encoded by different rhadinoviruses have been studied extensively. These studies revealed that the ORF73 expression product (pORF73) is a multifunctional protein essential for latency ... [more ▼]

ORF73 orthologues encoded by different rhadinoviruses have been studied extensively. These studies revealed that the ORF73 expression product (pORF73) is a multifunctional protein essential for latency that enables episome tethering to mitotic chromosomes and modulates cellular pathways implicated in growth and survival of latently infected cells. Comparison of pORF73 orthologues encoded by rhadinoviruses reveals important variations in amino acid sequence length and composition. Bovine herpesvirus 4 (BoHV-4) encodes by far the shortest ORF73 orthologue with a size equivalent to only 22% of the largest orthologues. The present study focused on determining if BoHV 4 ORF73 is a bona fide gene and investigating whether it is essential for latency as established for larger ORF73 orthologues. Our results demonstrate that BoHV-4 ORF73 is transcribed as immediate-early bicistronic mRNA together with ORF71. Using a BoHV-4 bacterial artificial chromosome clone, we produced a strain deleted for ORF73 and a revertant strain. Deletion of BoHV-4 ORF73 did not affect the capacity of the virus to replicate in vitro, but it prevented latent infection in vivo using a rabbit model. Interestingly, the strain deleted for ORF73 induced an anti-BoHV-4 humoral immune response comparable to that elicited by wild-type and revertant recombinants. Together, these results demonstrate that despite its relatively small size, BoHV-4 ORF73 is a functional homologue of larger rhadinovirus ORF73 orthologues, and highlight the potential of ORF73 deletion for the development of BoHV-4 as a vector in vaccinology. [less ▲]

Malignant catarrhal fever induced by alcelaphine herpesvirus 1 is associated with proliferation of CD8+ T cells supporting a latent infection

Poster (2009, September 11)

Alcelaphine herpesvirus 1 (AlHV 1), carried by wildebeest asymptomatically, causes malignant catarrhal fever (WD MCF) when cross species transmitted to a variety of susceptible species of the Artiodactyla ... [more ▼]

Alcelaphine herpesvirus 1 (AlHV 1), carried by wildebeest asymptomatically, causes malignant catarrhal fever (WD MCF) when cross species transmitted to a variety of susceptible species of the Artiodactyla order. Experimentally, WD-MCF can be induced in rabbits. The lesions observed are very similar to those described in natural host species. Here, we used the rabbit model and in vivo 5-Bromo-2’-Deoxyuridine (BrdU) incorporation to study WD-MCF pathogenesis. The results obtained can be summarized as follows. (i) AlHV-1 infection induces CD8+ T cell proliferation detectable as early as 15 days post-inoculation. (ii) While the viral load in peripheral blood mononuclear cells remains below the detection level during most of the incubation period, it increases drastically few days before death. At that time, at least 10% of CD8+ cells carry the viral genome; while CD11b+, IgM+ and CD4+ cells do not. (iii) RT-PCR analyses of mononuclear cells isolated from the spleen and the popliteal lymph node of infected rabbits revealed no expression of ORF25 and ORF9, low or no expression of ORF50, and high or no expression of ORF73. Based on these data, we propose a new model for the pathogenesis of WD-MCF. This model relies on proliferation of infected CD8+ cells supporting a predominantly latent infection. [less ▲]

Malignant catarrhal fever induced by alcelaphine herpesvirus 1 is associated with proliferation of CD8+ T cells supporting a latent infection

Conference (2009, April)

Alcelaphine herpesvirus 1 (AlHV 1), carried by wildebeest asymptomatically, causes malignant catarrhal fever (WD MCF) when cross species transmitted to a variety of susceptible species of the Artiodactyla ... [more ▼]

Alcelaphine herpesvirus 1 (AlHV 1), carried by wildebeest asymptomatically, causes malignant catarrhal fever (WD MCF) when cross species transmitted to a variety of susceptible species of the Artiodactyla order. Experimentally, WD-MCF can be induced in rabbits. The lesions observed are very similar to those described in natural host species. Here, we used the rabbit model and in vivo 5-Bromo-2’-Deoxyuridine (BrdU) incorporation to study WD-MCF pathogenesis. The results obtained can be summarized as follows. (i) AlHV-1 infection induces CD8+ T cell proliferation detectable as early as 15 days post-inoculation. (ii) While the viral load in peripheral blood mononuclear cells remains below the detection level during most of the incubation period, it increases drastically few days before death. At that time, at least 10% of CD8+ cells carry the viral genome; while CD11b+, IgM+ and CD4+ cells do not. (iii) RT-PCR analyses of mononuclear cells isolated from the spleen and the popliteal lymph node of infected rabbits revealed no expression of ORF25 and ORF9, low or no expression of ORF50, and high or no expression of ORF73. Based on these data, we propose a new model for the pathogenesis of WD-MCF. This model relies on proliferation of infected CD8+ cells supporting a predominantly latent infection. [less ▲]

Lung interstitial macrophages alter dendritic cell functions to prevent airway allergy in mice

in Journal of Clinical Investigation (2009), 119(12), 3723-38

The respiratory tract is continuously exposed to both innocuous airborne antigens and immunostimulatory molecules of microbial origin, such as LPS. At low concentrations, airborne LPS can induce a lung DC ... [more ▼]

The respiratory tract is continuously exposed to both innocuous airborne antigens and immunostimulatory molecules of microbial origin, such as LPS. At low concentrations, airborne LPS can induce a lung DC-driven Th2 cell response to harmless inhaled antigens, thereby promoting allergic asthma. However, only a small fraction of people exposed to environmental LPS develop allergic asthma. What prevents most people from mounting a lung DC-driven Th2 response upon exposure to LPS is not understood. Here we have shown that lung interstitial macrophages (IMs), a cell population with no previously described in vivo function, prevent induction of a Th2 response in mice challenged with LPS and an experimental harmless airborne antigen. IMs, but not alveolar macrophages, were found to produce high levels of IL-10 and to inhibit LPS-induced maturation and migration of DCs loaded with the experimental harmless airborne antigen in an IL-10-dependent manner. We further demonstrated that specific in vivo elimination of IMs led to overt asthmatic reactions to innocuous airborne antigens inhaled with low doses of LPS. This study has revealed a crucial role for IMs in maintaining immune homeostasis in the respiratory tract and provides an explanation for the paradox that although airborne LPS has the ability to promote the induction of Th2 responses by lung DCs, it does not provoke airway allergy under normal conditions. [less ▲]