Ex Vivo Bioluminescence Detection of Alcelaphine Herpesvirus 1 Infection during Malignant Catarrhal Fever

Abstract

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 is 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 nonlymphoid 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 rabbits infected with the parental wild-type strain, with hyperthermia and increases of both CD8 ؉ T cell 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 nonlymphoid organs are mainly CD8 ؉ T cells and that latency is predominant during WD-MCF.

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

“…Although no function could be attributed to this region, it might be involved in the pathogenesis of MCF. Our previous results in the rabbit model showed the low or absence of productive virus infection in MCF (14,15), and we confirmed and extended these results in a whole-genome approach in calves. However, it remained unclear whether AlHV-1 persistence was mediated by classical episomal maintenance in proliferating cells or rather, explained, for instance, by a succession of abortive lytic cycles.…”

“…Absence of virus growth defect in culture was in line with some reports showing that γ-herpesvirus ORF73 is not essential for virus replication in vitro (21)(22)(23)(24), although MuHV-4 or Rhesus monkey rhadinovirus showed lytic replication deficit under certain conditions or enhancement, respectively (25,26). To further address the effect of ORF73 deletion on virus replication in vivo, we used the luciferase + WT AlHV-1 (247N-luc + ) BAC plasmid (15) to produce a 247N-luc + -73 null recombinant virus (SI Appendix, Fig. S11).…”

“…Initially, the very low levels of detection of infected cells in lesions led to the hypothesis that MCF could be caused by very few infected cells interacting with the surrounding uninfected T cells, resulting in their deregulation (12,13). However, recent reports have suggested that virus infection in vivo might be more frequent than previously thought (14,15). MCF can be experimentally induced in rabbits, where the observed lesions are indistinguishable from the lesions described in the MCF-susceptible species (16).…”

“…Other than the proliferation of CD8 + T cells in lymphoid tissues, MCF is characterized by the infiltration of activated and cytotoxic CD3 + CD8 + CD4 − T cells in the perivascular spaces of all tissues and organs (17). Using a recombinant virus strain of AlHV-1 expressing the firefly luciferase, we recently showed that the macroscopic distribution of AlHV-1 infection in explanted organs of MCF-developing rabbits colocalizes with the distribution of lesions in both lymphoid and nonlymphoid tissues of MCF-developing rabbits (15). The lack of detection of infectious viral particles together with the low (or no) expression of few selected viral genes normally expressed during productive viral infection suggested the absence or rarity of cells supporting productive infection in the tissues (14,15).…”

“…Using a recombinant virus strain of AlHV-1 expressing the firefly luciferase, we recently showed that the macroscopic distribution of AlHV-1 infection in explanted organs of MCF-developing rabbits colocalizes with the distribution of lesions in both lymphoid and nonlymphoid tissues of MCF-developing rabbits (15). The lack of detection of infectious viral particles together with the low (or no) expression of few selected viral genes normally expressed during productive viral infection suggested the absence or rarity of cells supporting productive infection in the tissues (14,15). These results suggested that AlHV-1 infection might not be productive during MCF and could be latent.…”