Wildebeest-Derived Malignant Catarrhal Fever: A Bovine Peripheral T Cell Lymphoma Caused by Cross-Species Transmission of Alcelaphine Gammaherpesvirus 1.

Gammaherpesviruses (γHVs) include viruses that can induce lymphoproliferative diseases and tumors. These viruses can persist in the long term in the absence of any pathological manifestation in their natural host. Alcelaphine gammaherpesvirus 1 (AlHV-1) belongs to the genus Macavirus and asymptomatically infects its natural host, the wildebeest (Connochaetes spp.). However, when transmitted to several susceptible species belonging to the order Artiodactyla, AlHV-1 is responsible for the induction of a lethal lymphoproliferative disease, named wildebeest-derived malignant catarrhal fever (WD-MCF). Understanding the pathogenic mechanisms responsible for the induction of WD-MCF is important to better control the risks of transmission and disease development in susceptible species. The aim of this review is to synthesize the current knowledge on WD-MCF with a particular focus on the mechanisms by which AlHV-1 induces the disease. We discuss the potential mechanisms of pathogenesis from viral entry into the host to the maintenance of viral genomes in infected CD8+ T lymphocytes, and we present current hypotheses to explain how AlHV-1 infection induces a peripheral T cell lymphoma-like disease.

Alcelaphine herpesvirus 1 genes A7 and A8 regulate viral spread and are essential for malignant catarrhal fever.

Alcelaphine herpesvirus 1 (AlHV-1) is a gammaherpesvirus that is carried asymptomatically by wildebeest. Upon cross-species transmission to other ruminants, including domestic cattle, AlHV-1 induces malignant catarrhal fever (MCF), which is a fatal lymphoproliferative disease resulting from proliferation and uncontrolled activation of latently infected CD8+ T cells. Two laboratory strains of AlHV-1 are used commonly in research: C500, which is pathogenic, and WC11, which has been attenuated by long-term maintenance in cell culture. The published genome sequence of a WC11 seed stock from a German laboratory revealed the deletion of two major regions. The sequence of a WC11 seed stock used in our laboratory also bears these deletions and, in addition, the duplication of an internal sequence in the terminal region. The larger of the two deletions has resulted in the absence of gene A7 and a large portion of gene A8. These genes are positional orthologs of the Epstein-Barr virus genes encoding envelope glycoproteins gp42 and gp350, respectively, which are involved in viral propagation and switching of cell tropism. To investigate the degree to which the absence of A7 and A8 participates in WC11 attenuation, recombinant viruses lacking these individual functions were generated in C500. Using bovine nasal turbinate and embryonic lung cell lines, increased cell-free viral propagation and impaired syncytia formation were observed in the absence of A7, whereas cell-free viral spread was inhibited in the absence of A8. Therefore, A7 appears to be involved in cell-to-cell viral spread, and A8 in viral cell-free propagation. Finally, infection of rabbits with either mutant did not induce the signs of MCF or the expansion of infected CD8+ T cells. These results demonstrate that A7 and A8 are both essential for regulating viral spread and suggest that AlHV-1 requires both genes to efficiently spread in vivo and reach CD8+ T lymphocytes and induce MCF.

Seroprevalence of malignant catarrhal fever virus in captive wildebeest (Connochaetes sp.) in France.

Alcelaphine herpesvirus 1 (AlHV-1) is a gammaherpesvirus carried asymptomatically by wildebeests (Connochaetes sp.) in sub-Saharan Africa. Although asymptomatic in wildebeest, AlHV-1 infection in a number of other ruminant species causes a severe and fatal lymphoproliferative disease named wildebeest-derived malignant catarrhal fever (WD-MCF). Several endangered species of captive ruminants are highly susceptible to developing WD-MCF if infected by AlHV-1, which is a critical concern in zoos, game reserves and wildlife parks where wildebeests are also kept in captivity. Here, we investigated the seroprevalence of AlHV-1 in 52 captive wildebeests randomly sampled from five different zoos in France. We found 46% (24/52) seropositive animals and detected AlHV-1 DNA in one of them, demonstrating that AlHV-1 infection is present in captive wildebeests in France. In an interesting manner, the repartition of seropositive wildebeests was not homogenous between zoos with 100% (20/20) of seronegative animals in three parks. These results further highlight the importance of considering WD-MCF as a threat for clinically susceptible species and encourage for testing AlHV-1 infection in captive wildebeests as a management control strategy.

Macavirus latency-associated protein evades immune detection through regulation of protein synthesis in cis depending upon its glycin/glutamate-rich domain.

Alcelaphine herpesvirus 1 (AlHV-1) is a γ-herpesvirus (γ-HV) belonging to the macavirus genus that persistently infects its natural host, the wildebeest, without inducing any clinical sign. However, cross-transmission to other ruminant species causes a deadly lymphoproliferative disease named malignant catarrhal fever (MCF). AlHV-1 ORF73 encodes the latency-associated nuclear antigen (LANA)-homolog protein (aLANA). Recently, aLANA has been shown to be essential for viral persistence in vivo and induction of MCF, suggesting that aLANA shares key properties of other γ-HV genome maintenance proteins. Here we have investigated the evasion of the immune response by aLANA. We found that a glycin/glutamate (GE)-rich repeat domain was sufficient to inhibit in cis the presentation of an epitope linked to aLANA. Although antigen presentation in absence of GE was dependent upon proteasomal degradation of aLANA, a lack of GE did not affect protein turnover. However, protein self-synthesis de novo was downregulated by aLANA GE, a mechanism directly associated with reduced antigen presentation in vitro. Importantly, codon-modification of aLANA GE resulted in increased antigen presentation in vitro and enhanced induction of antigen-specific CD8+ T cell responses in vivo, indicating that mRNA constraints in GE rather than peptidic sequence are responsible for cis-limitation of antigen presentation. Nonetheless, GE-mediated limitation of antigen presentation in cis of aLANA was dispensable during MCF as rabbits developed the disease after virus infection irrespective of the expression of full-length or GE-deficient aLANA. Altogether, we provide evidence that inhibition in cis of protein synthesis through GE is likely involved in long-term immune evasion of AlHV-1 latent persistence in the wildebeest natural host, but dispensable in MCF pathogenesis.

Genomic duplication and translocation of reactivation transactivator and bZIP-homolog genes is a conserved event in alcelaphine herpesvirus 1.

Alcelaphine herpesvirus 1 (AlHV-1) is a gammaherpesvirus carried asymptomatically by wildebeest. Upon cross-species transmission, AlHV-1 induces malignant catarrhal fever (MCF), a fatal lymphoproliferative disease of ruminants, including cattle. The strain C500 has been cloned as an infectious, pathogenic bacterial artificial chromosome (BAC) that is used to study MCF. Although AlHV-1 infection can be established in cell culture, multiple passages in vitro cause a loss of virulence associated with rearrangements of the viral genome. Here, sequencing of the BAC clone showed that the long unique region (LUR) of the genome is nearly identical to that of the previously sequenced strain from which the BAC was derived, and identified the duplication and translocation of a region from within LUR, containing the entire coding sequences of ORF50-encoding reactivation transactivator Rta and A6-encoding bZIP protein genes. The duplicated region was further located to a position within the terminal repeat (TR) and its deletion resulted in lower ORF50 expression levels and reduced viral fitness. Finally, the presence of a similar but not identical duplication and translocation containing both genes was found in AlHV-1 strain WC11. These results indicate that selection pressure for enhanced viral fitness may drive the duplication of ORF50 and A6 in AlHV-1.

Small RNA deep sequencing identifies viral microRNAs during malignant catarrhal fever induced by alcelaphine herpesvirus 1.

Alcelaphine herpesvirus 1 (AlHV-1) is a c-herpesvirus (c-HV) carried asymptomatically by wildebeest. Upon cross-species transmission, AlHV-1 induces a fatal lymphoproliferative disease named malignant catarrhal fever (MCF) in many ruminants, including cattle, and the rabbit model. Latency has been shown to be essential for MCF induction. However, the mechanisms causing the activation and proliferation of infected CD8+T cells are unknown. Many c-HVs express microRNAs (miRNAs). These small non-coding RNAs can regulate expression of host or viral target genes involved in various pathways and are thought to facilitate viral infection and/or mediate activation and proliferation of infected lymphocytes. The AlHV-1 genome has been predicted to encode a large number of miRNAs. However, their precise contribution in viral infection and pathogenesis in vivo remains unknown. Here, using cloning and sequencing of small RNAs we identified 36 potential miRNAs expressed in a lymphoblastoid cell line propagated from a calf infected with AlHV-1 and developing MCF. Among the sequenced candidate miRNAs, 32 were expressed on the reverse strand of the genome in two main clusters. The expression of these 32 viral miRNAs was further validated using Northern blot and quantitative reverse transcription PCR in lymphoid organs of MCF developing calves or rabbits. To determine the concerted contribution in MCF of 28 viralmiRNAs clustered in the non-protein-coding region of the AlHV-1 genome, a recombinant virus was produced. The absence of these 28 miRNAs did not affect viral growth in vitro or MCF induction in rabbits, indicating that the AlHV-1 miRNAs clustered in this non-protein-coding genomic region are dispensable for MCF induction.

Viral semaphorin inhibits dendritic cell phagocytosis and migration but is not essential for gammaherpesvirus-induced lymphoproliferation in malignant catarrhal fever.

UNLABELLED: Viral semaphorins are semaphorin 7A (sema7A) mimics found in pox- and herpesviruses. Among herpesviruses, semaphorins are encoded by gammaherpesviruses of the Macavirus genus only. Alcelaphine herpesvirus 1 (AlHV-1) is a macavirus that persistently infects wildebeest asymptomatically but induces malignant catarrhal fever (MCF) when transmitted to several species of susceptible ruminants and the rabbit model. MCF is caused by the activation/proliferation of latently infected T lymphocytes. Viral semaphorins have been suggested to mediate immune evasion mechanisms and/or directly alter host T cell function. We studied AlHV-sema, the viral semaphorin encoded by the A3 gene of AlHV-1. Phylogenetic analyses revealed independent acquisition of pox- and herpesvirus semaphorins, suggesting that these proteins might have distinct functions. AlHV-sema showed a predicted three-dimensional structure very similar to sema7A and conserved key residues in sema7A-plexinC1 interaction. Expression analyses revealed that AlHV-sema is a secreted 93-kDa glycoprotein expressed during the early phase of virus replication. Purified AlHV-sema was able to bind to fibroblasts and dendritic cells and induce F-actin condensation and cell retraction through a plexinC1 and Rho/cofilin-dependent mechanism. Cytoskeleton rearrangement was further associated with inhibition of phagocytosis by dendritic cells and migration to the draining lymph node. Next, we generated recombinant viruses and demonstrated that the lack of A3 did not significantly affect virus growth in vitro and did not impair MCF induction and associated lymphoproliferative lesions. In conclusion, AlHV-sema has immune evasion functions through mechanisms similar to poxvirus semaphorin but is not directly involved in host T cell activation during MCF. IMPORTANCE: Whereas most poxviruses encode viral semaphorins, semaphorin-like genes have only been identified in few gammaherpesviruses belonging to the Macavirus genus. Alcelaphine herpesvirus 1 (AlHV-1) is a macavirus carried asymptomatically by wildebeest but induces a latency-associated lymphoproliferative disease of T lymphocytes in various ruminant species, namely, malignant catarrhal fever (MCF). Viral semaphorins have been hypothesized to have immune evasion functions and/or be involved in activating latently infected T cells. We present evidence that the viral semaphorin AlHV-sema inhibits dendritic cell phagocytosis and migration to the draining lymph node, both being indispensable mechanisms for protective antiviral responses. Next, we engineered recombinant viruses unable to express AlHV-sema and demonstrated that this protein is dispensable for the induction of MCF. In conclusion, this study suggests that herpesvirus and poxvirus semaphorins have independently evolved similar functions to thwart the immune system of the host while AlHV-sema is not directly involved in MCF-associated T-cell activation.

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.

La forme africaine du coryza gangreneux est une maladie lymphoproliférative létale causée par l'herpèsvirus alcélaphin 1.

Gammaherpesviruses include viruses able to induce lymphoproliferative diseases and tumors. These viruses can also establish a long and persistent asymptomatic infection in their natural host. Among gammaherpesviruses, AlHV-1 belongs to the Macavirus genus and infects asymptomatically its natural host, the wildebeest (Connochaetes spp.). However, this virus induces a lethal lymphoproliferative disease named the African form of malignant catarrhal fever (MCF) when transmitted to a large number of susceptible species belonging to the Artiodactyla order. Control of MCF in regions where AlHV-1 is endemic is of great importance and directly depends on the understanding of the pathogenic mechanisms responsible for the induction of MCF. The goal of the present review is to summarize current knowledge on AlHV-1 and MCF with a particular interest on the mechanisms used by AlHV-1 to induce the disease. Amongst different models of pathogenesis, the particular role of viral latency will be addressed based on current data. Finally, the evolutionary relationship between the wildebeest, AlHV-1 and the susceptible species to MCF will be discussed.

Ex vivo bioluminescence detection of alcelaphine herpesvirus 1 infection during malignant catarrhal fever.

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.