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.Peer reviewe

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

Study of the viral semaphorin encoded by the A3 gene of alcelaphine herpesvirus 1

Among gammaherpesvirus, the Macavirus genus is composed of viruses associated to malignant catarrhal fever (MCF) and other phylogenetically related viruses. MCF is a frequently fatal lymphoproliferative disease. Three macaviruses inducing MCF have been entirely sequenced: alcelaphine herpesvirus 2 (AlHV-2), ovine herpesvirus 2 (OvHV-2) and alcelaphine herpesvirus 1 (AlHV-1). Sheep carries OvHV-2 asymptomatically while wildebeest is infected with AlHV-1 without developing any clinical signs or lesions. Both viruses represent the most studied macaviruses. In susceptible ruminants, OvHV-2 and AlHV-1 induce the sheep-associated form and the wildebeest-derived form of MCF (WD-MCF), respectively. Economic consequences of WD-MCF are significant in sub-Saharan Africa. WD-MCF is characterized by the proliferation and infiltration of lymphoblastoid T cells surrounding blood vessels and can be considered as a model for peripheral T cell lymphoma caused by a virus. Rabbits are used as experimental model to study MCF. This species develops clinical signs and lesions that they are indistinguishable from those observed in other susceptible species. Until recently, available data on WD-MCF pathogenesis were limited to the simple description clinical signs and lesions. Recently, it was demonstrated that CD8+ T cells proliferate and that this cellular expansion is associated with a severe increase of the viral load in PBMC and lymphoid organs (Dewals et al., 2008). The cloning of the AlHV-1 genome as an infectious and pathogenic bacterial artificial chromosome (BAC) has greatly facilitated the study of individual gene of AlHV-1 (Dewals et al., 2006). Among herpesviruses, viral semaphorins can only be found in members of the Macavirus genus. OvHV 2 encodes Ov3, and AlHV-2 and AlHV-1 encode A3, both genes encoding a semaphorin homolog. Semaphorins are proteins characterized by a conserved amino-terminal domain, the SEMA domain. The roles of the semaphorins on cytoskeleton dynamics have been widely studied. Viral semaphorins could mediate immune evasion mechanisms or viral dissemination and could be involved in specific properties of macaviruses. The first study of the present thesis was dedicated to the investigation of the pathogenesis of WD-MCF and the role of latency. We investigated the distribution of the AlHV-1 infection in the lesions and demonstrated that the infiltration of CD8+ T cells in different lymphoid and non-lymphoid organs and tissues is directly associated with a non-productive viral infection. The second study focused on the A3 gene of AlHV-1 and its potential functions during WD-MCF. We showed that the A3 gene is expressed during the early phase of the viral infection and encodes a functional semaphorin that was termed AlHV-sema. AlHV-sema was able to induce cell retraction. Despite the observed independent acquisition of pox- and herpesvirus semaphorins, AlHV-sema inhibited phagocytosis by dendritic cells and migration to the draining lymph node through mechanisms similar to poxvirus semaphorin. AlHV-sema could also facilitate viral dissemination or confer immune evasion functions. Next, we investigated whether AlHV-sema could affect WD-MCF induction. We did not observe any effect of the absence of AlHV-sema expression during the development of WD-MCF after rabbit nasal infection. Macaviruses are swine and ruminant gammaherpesviruses responsible for a latent asymptomatic infection in their natural species. The development of MCF in other ruminant susceptible species is due to cross-species transmission. During evolution, the gene selection in susceptible species is certainly reduced due to the fact that these species are dead-end hosts. Thus, it is difficult to address the role of AlHV-1 specific genes in MCF as these genes have evolved in other species. Nevertheless, we brought in this work important insight for our understanding of the pathogenesis of WD-MCF and we identified AlHV-sema as a potential immunoevasion factor. DEWALS B., MYSTER F., PALMEIRA L., GILLET L., ACKERMANN M., VANDERPLASSCHEN A. Ex vivo bioluminescence detection of alcelaphine herpesvirus 1 infection during malignant catarrhal fever. J. Virol., 2011, 85, 6941-6954. MYSTER F., PALMEIRA L., SOREL O., BOUILLENNE F., DEPAUW E., SCHWARTZ-CORNIL I., VANDERPLASSCHEN A., DEWALS B.G. Viral semaphorin inhibits dendritic cell phagocytosis and migration but is not essential for gamma-herpesvirus-induced lymphoproliferation in malignant catarrhal fever. J. Virol., 2015

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

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

A7 gene expression is essential for alcelaphine herpesvirus 1-driven CD8+ T cell expansion and typical malignant catarrhal fever

peer reviewedAlcelaphine herpesvirus 1 (AlHV-1) persists in wildebeest asymptomatically but induces malignant catarrhal fever (MCF), a fatal lymphoproliferative disease upon transmission to several ruminants, including cattle. Our recent data have demonstrated that AlHV-1 latently infects CD8+ T-cells resulting in their proliferation and MCF lesions. How AlHV-1 infects CD8+ T-cells and induce their proliferation remain however unknown. To better understand MCF pathogenesis, we first used Illumina high-throughput sequencing to obtain the full genomic sequence of the highly passaged attenuated strain WC11. Sequencing data revealed a genomic sequence very similar to the pathogenic strain C500 with only four ORFs that were not conserved in WC11, including full deletion of the gene A7. A7 is a positional homolog of Epstein-Barr virus (EBV) BZLF2 encoding the C-type lectin-like glycoprotein gp42. Gp42 is expressed in the envelope of EBV virions and mediates entry into B cells. Likewise, A7 has been predicted to encode a glycoprotein containing a C-type lectin-like domain. The absence of A7 in the high passaged attenuated strain could participate in the attenuation of WC11 through a defect of virus entry into target cells. Hence, we used the C500 BAC clone to generate an A7STOP recombinant strain and observed that a lack of A7 expression resulted in significant increased viral growth in fibroblasts in vitro. Next, we infected rabbits intranasally with the A7STOP virus. While the A7STOP virus induced hyperthermia with a significant delay compared to the WT virus, neither enlargement of lymphoid organs nor typical expansion of CD8+ T cells could be detected in the absence of A7 expression. Interestingly, viral DNA loads and antibody responses were significantly reduced in lymphoid tissues of A7STOP infected animals. Finally, the infiltrations of lymphoid cells typically observed in WT-infected animals were severely reduced in absence of A7. In conclusion, these findings demonstrated that the lack of A7 significantly alters the development of MCF, likely through deviating CD8+ T cell tropism

Ex vivo bioluminescent detection of alcelaphine herpesvirus 1 infection during malignant catarrhal fever induced in rabbits

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. The lesions observed are very similar to those described in natural host species. Recently, we demonstrated that WD-MCF induced by AlHV-1 in rabbits is associated with the proliferation of CD8+ cells supporting a latent type of infection. In the present study, we investigated whether the virus could be detected ex vivo in the tissues of rabbits developing WD-MCF. Taking advantage of the recent cloning of the AlHV-1 genome as a bacterial artificial chromosome (BAC), we produced a recombinant plasmid encoding a firefly luciferase (LUC) expression cassette inserted in a non-coding region of the AlHV-1 genome. In vitro, the reconstituted AlHV-1 LUC strain replicated comparably to the parental strain in permissive cells and was able to induce a bioluminescent signal. In vivo, rabbits infected with the AlHV-1 LUC strain developed WD-MCF similarly to the parental wild-type strain with hyperthermia, increased CD8/CD4 ratio and viral genomic charge over time in PBMC and in lymph nodes at time of death. To identify the presence of AlHV-1 infection ex vivo, various organs of infected rabbits developing WD-MCF were analysed by bioluminescent imaging. Luciferase activity could be detected macroscopically at the time of death in most of analyzed organs including lung, popliteal and mesenteric lymph nodes, spleen, liver, kidney and appendix. Infectious virus could be isolated following co-cultures of lymph node and permissive cells, and the isolated virus retained the ability to induce a bioluminescent signal. In conclusion, we produced an AlHV-1 LUC recombinant and we were able to detect the AlHV-1 infection ex vivo in many organs at the time of death

Cis-acting inhibition of MHC class I-restricted epitope presentation by Alcelaphine herpesvirus 1 genome maintenance protein

γ-Herpesviruses persist as latent episomes in actively dividing lymphocytes. Their consequent need to express a viral genome maintenance protein (GMP) during latency presents a potential immune target. However, the GMPs from several γ-herpesviruses have evolved related strategies to limit their own MHC class I epitope presentation to cytotoxic T lymphocytes (CTLs). Alcelaphine herpesvirus 1 (AlHV-1) is a γ-herpesvirus that persists asymptomatically in its natural host, the wildebeest. However, AlHV-1 transmission to a large number of susceptible ruminants, including cattle, results in the development of a lethal lymphoproliferative disease named malignant catarrhal fever (MCF). We recently observed that the AlHV-1 GMP-homologue encoded by ORF73 is highly expressed during MCF and that the impairment of its expression renders AlHV-1 unable to induce MCF. With its 1300 aa, AlHV-1 ORF73 is the largest γ-herpesvirus GMP described to date and contains a large acidic internal repeat region that could be involved in the cis-acting CTL evasion mechanism. Here, we sought to determine the CTL evasion properties of AlHV-1 ORF73. We first performed bioinformatic analyses to characterize the protein domains. Then, we used an in vitro assay to demonstrate that ORF73 severely limits the presentation at the cell surface of an MHC class I-restricted epitope linked to ORF73 in cis. These results suggest that AlHV-1 has developed mechanisms to evade cytotoxic anti-viral response during latency. The exact mechanisms explaining the presentation defect remain to be deciphered as well as the role of the cis-acting CTL evasion mechanism of ORF73 in the pathogenesis of MCF

A7 gene expression is essential for alcelaphine herpesvirus 1-driven CD8+ T cell expansion and typical malignant catarrhal fever.

peer reviewedAlcelaphine herpesvirus 1 (AlHV-1) persists in wildebeest asymptomatically but induces malignant catarrhal fever (MCF), a fatal lymphoproliferative disease upon transmission to several ruminants, including cattle. Our recent data have demonstrated that AlHV-1 latently infects CD8+ T-cells resulting in their proliferation and MCF lesions. How AlHV-1 infects CD8+ T-cells and induce their proliferation remain however unknown. To better understand MCF pathogenesis, we first used Illumina high-throughput sequencing to obtain the full genomic sequence of the highly passaged attenuated strain WC11. Sequencing data revealed a genomic sequence very similar to the pathogenic strain C500 with only four ORFs that were not conserved in WC11, including full deletion of the gene A7. A7 is a positional homolog of Epstein-Barr virus (EBV) BZLF2 encoding the C-type lectin-like glycoprotein gp42. Gp42 is expressed in the envelope of EBV virions and mediates entry into B cells. Likewise, A7 has been predicted to encode a glycoprotein containing a C-type lectin-like domain. The absence of A7 in the high passaged attenuated strain could participate in the attenuation of WC11 through a defect of virus entry into target cells. Hence, we used the C500 BAC clone to generate an A7STOP recombinant strain and observed that a lack of A7 expression resulted in significant increased viral growth in fibroblasts in vitro. Next, we infected rabbits intranasally with the A7STOP virus. While the A7STOP virus induced hyperthermia with a significant delay compared to the WT virus, neither enlargement of lymphoid organs nor typical expansion of CD8+ T cells could be detected in the absence of A7 expression. Interestingly, viral DNA loads and antibody responses were significantly reduced in lymphoid tissues of A7STOP infected animals. Finally, the infiltrations of lymphoid cells typically observed in WT-infected animals were severely reduced in absence of A7. In conclusion, these findings demonstrated that the lack of A7 significantly alters the development of MCF, likely through deviating CD8+ T cell tropism

Self-inhibition of synthesis reduces antigen presentation of the alcelaphine herpesvirus 1-encoded latency-associated protein, aLANA

Alcelalphine herpesvirus 1 (AlHV-1) persistently infects its natural host, the wildebeest, without inducing any clinical signs. However, cross-transmission to other ruminant species leads to the development of a deadly lymphoproliferative disease named malignant catarrhal fever (MCF). AlHV-1 ORF73 encodes the latency-associated nuclear antigen (LANA)-homolog protein termed aLANA. Similarly to other viral genome maintenance proteins encoded by gammaherpesviruses, aLANA has recently been shown to be essential for viral persistence and induction of MCF. Here we have investigated the self-inhibition of antigen presentation by aLANA and the potential role of such mechanism during the development of MCF. We showed that the GE-rich repeat domain of aLANA was sufficient to inhibit the presentation of an epitope linked to it. Though antigen presentation in absence of GE was dependent upon proteasomal degradation of aLANA, a lack of GE did not affect protein turnover. We further found that similarly to EBNA-1 GAr, aLANA GE downregulated protein self-synthesis. Likewise, such mechanism could be associated with reduced antigen presentation in vitro. In addition, in-frame insertion of GE repeat domain in a heterologous eGFP protein significantly down-regulated protein steady-state levels and self-antigen presentation. Next, we modified the AlHV-1 ORF73 gene sequence to reduce the purine bias in GE, without affecting the peptidic sequence. Such codon-modified aLANA GEm construct displayed increased antigen presentation. Finally, we generated an AlHV-1 recombinant strain expressing a GE-deficient aLANA protein and observed that viral growth was not affected in vitro by the absence of aLANA GE domain and MCF could be induced in rabbits irrespective of the expression of full-length aLANA or GE-deficient aLANA protein

The A3 gene of Alcelaphine herpesvirus 1 encodes a viral semaphorin that is non-essential for the induction of malignant catarrhal fever

Alcelaphine herpesvirus 1 (AlHV-1) is a γ-Herpesvirus 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 frequently fatal lymphoproliferative and degenerative disease of ruminants. Experimentally, WD-MCF can be reproduced in rabbits. The A3 open reading frame (ORF) of the AlHV-1 encodes a putative semaphorin homolog protein, thereafter named AlHV-sema. Semaphorins are secreted and membrane-associated proteins characterized by a conserved amino-terminal ‘Sema’ domain. Initially identified as guidance factors that assist axons pathfinding during neural development, semaphorins have been shown over the last decade to have significant functions in various processes of immunoregulation. Bioinformatics analyses revealed that AlHV-sema has a high homology to the cellular Sema7A. Besides its roles in neural development, Sema7A has been shown to play pivotal functions in the regulation of cytokine secretion and as a tumor suppressor. In order to investigate whether AlHV-Sema could play a role in the pathogenesis of WD-MCF, 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 similarly to that observed with the parental strain with both severely increased CD8+ T cell frequencies and viral genomic charge over time in peripheral blood and in lymph nodes at time of death, as well as indistinguishable histopathological lesions in lymphoid organs and in liver, lung and kidney. In conclusion, this study demonstrates that AlHV-sema is not essential for the induction of WD-MCF in rabbits