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

Leonor Palmeiraa, Océane Sorela, Willem Van Campeb, Christel Boudrya, Stefan Roelsb, Françoise Mystera, Anca Reschnera, Pierre G. Couliec, Pierre Kerkhofsb, Alain Vanderplasschena, and Benjamin G. Dewalsa,1

aDepartment of Infectious and Parasitic Diseases, Immunology-Vaccinology (B43b), Faculty of Veterinary Medicine, University of Liège, B-4000 Liège, Belgium; bVeterinary and Agrochemical Research Center (CODA-CERVA), B-1180 Brussels, Belgium; and cde Duve Institute, University of Louvain, B-1200 Brussels, Belgium

Edited by Bill Sugden, University of Wisconsin, Madison, WI, and accepted by the Editorial Board March 20, 2013 (received for review September 24, 2012) Wildebeests carry asymptomatically alcelaphine herpesvirus 1 estimated, with recent reports showing that MCF is perceived to (AlHV-1), a γ-herpesvirus inducing malignant catarrhal fever (MCF) be the cattle disease with the highest economical and social to several ruminant species (including cattle). This acute and lethal impacts in these areas (7, 8). In addition, MCF has been reported lymphoproliferative disease occurs after a prolonged asymptomatic throughout the world in game farms or zoological collections incubation period after transmission. Our recent findings with the where mixed ruminant species, including wildebeest, are kept (9). rabbit model indicated that AlHV-1 infection is not productive during The mechanisms responsible for the lymphoproliferative and MCF. Here, we investigated whether latency establishment could ex- degenerative lesions observed in MCF are unknown (3, 10, 11). plain this apparent absence of productive infection and sought to Initially, the very low levels of detection of infected cells in lesions determine its role in MCF pathogenesis. First, whole-genome cellular led to the hypothesis that MCF could be caused by very few and viral gene expression analyses were performed in lymph nodes of infected cells interacting with the surrounding uninfected T cells, MCF-developing calves. Whereas a severe disruption in cellular genes resulting in their deregulation (12, 13). However, recent reports was observed, only 10% of the entire AlHV-1 genome was expressed, have suggested that virus infection in vivo might be more frequent MICROBIOLOGY contrasting with the 45% observed during productive infection in than previously thought (14, 15). MCF can be experimentally in- vitro. In vivo, the expressed viral genes included the latency-associated duced in rabbits, where the observed lesions are indistinguishable nuclear antigen homolog ORF73 but none of the regions known to from the lesions described in the MCF-susceptible species (16). be essential for productive infection. Next, genomic conformation Using this model, we have recently shown that AlHV-1 infection is analyses revealed that AlHV-1 was essentially episomal, further sug- responsible for the induction of a severe proliferation of CD8+ gesting that MCF might be the consequence of a latent infection T cells in peripheral blood mononuclear cells (PBMCs) and lym- rather than abortive lytic infection. This hypothesis was further + phoid organs (14). It has also been shown that the infection is re- supported by the high frequencies of infected CD8 T cells during stricted to CD8+ cells in PBMCs and that at least 10% of these MCF using immunodetection of ORF73 protein and single-cell RT-PCR cells in PBMCs contain the viral genome. Other than the pro- approaches. Finally, the role of latency-associated ORF73 was ad- liferation of CD8+ T cells in lymphoid tissues, MCF is character- dressed. A lack of ORF73 did not impair initial virus replication in − ized by the infiltration of activated and cytotoxic CD3+CD8+CD4 vivo, but it rendered AlHV-1 unable to induce MCF and persist in T cells in the perivascular spaces of all tissues and organs (17). vivo and conferred protection against a lethal challenge with a WT fi fl virus. Together, these findings suggest that a latent infection is Using a recombinant virus strain of AlHV-1 expressing the re y essential for MCF induction. luciferase, we recently showed that the macroscopic distribution of AlHV-1 infection in explanted organs of MCF-developing rabbits whole-genome transcriptomics | peripheral T-cell lymphoma colocalizes with the distribution of lesions in both lymphoid and nonlymphoid tissues of MCF-developing rabbits (15). The lack of Connochaetes taurinus detection of infectious viral particles together with the low (or no) bout 1.3 million wildebeests ( ) mi- expression of few selected viral genes normally expressed during Agrate over 1,800 miles in eastern Africa every year (1, 2). productive viral infection suggested the absence or rarity of cells Local farmers, like the Maasai people, are nomadic pastoralists supporting productive infection in the tissues (14, 15). These who center their lifestyle on livestock herding, mostly in grazing results suggested that AlHV-1 infection might not be productive areas shared with wildlife. The great annual spectacle of the during MCF and could be latent. wildebeest migration directly endangers the livelihood of the Here, we investigated whether latency establishment could local herders, because cattle are highly susceptible to a fatal viral explain the apparent absence of productive infection and exam- disease named malignant catarrhal fever (MCF), which is mainly ined the role of AlHV-1 latency in MCF pathogenesis. First, we transmitted by the 500,000 wildebeest calves born each year. MCF is an acute, sporadic, and fatal pansystemic lymphoproli- ferative disease of a variety of species of the Artiodactyla order, including cattle. The main causative agents of MCF are two γ-herpesviruses that have been recently grouped in the Macavirus Author contributions: A.V. and B.G.D. designed research; L.P., O.S., W.V.C., C.B., S.R., F.M., ovine herpesvirus 2 alcelaphine herpesvirus 1 A.R., and B.G.D. performed research; P.G.C. contributed new reagents/analytic tools; L.P., genus, (OvHV-2) and P.K., A.V., and B.G.D. analyzed data; and B.G.D. wrote the paper. (AlHV-1). These viruses cause no apparent disease in their natural The authors declare no conflict of interest. host species. Sheep are naturally infected by OvHV-2, which is This article is a PNAS Direct Submission. B.S. is a guest editor invited by the Editorial Board. responsible for the sporadic sheep-associated form of MCF. Data deposition: The data reported in this paper have been deposited in the Gene Ex- Wildebeests carry AlHV-1, responsible for the wildebeest-derived pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession nos. GSE40644 form of the disease (3, 4). The prevalence of AlHV-1 infection in and GPL16018). wildebeest is close to 100%, and transmission mainly occurs during 1To whom correspondence should be addressed. E-mail: [email protected]. fi – the calving period and in the rst 3 4 mo of life (5, 6). MCF impact This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. on the local pastoralist populations has largely been under- 1073/pnas.1216531110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1216531110 PNAS Early Edition | 1of10 Downloaded by guest on September 28, 2021 studied MCF in calves after experimental AlHV-1 infection and Among all annotated ORFs, only three ORFs were not associated used a whole-genome approach to analyze both cellular and viral with an expressed transcript (A5, ORF18, and ORF56). In addi- RNA expression in the lymph nodes of MCF-developing calves. tion to predicted ORFs, 23 additional expressed regions (ERs) Our findings strongly confirmed an absence of productive in- did not correspond to annotated ORFs (Fig. 1A and SI Appendix, fection in MCF together with a profound disruption of the cellular Table S1). Their roles during viral infection are unknown. As gene expression profile. Next, we examined the viral genomic a whole, these results not only experimentally confirmed 96% of conformation and revealed that AlHV-1 genomes are essentially viral annotated ORFs and identified 23 additional ER, but they maintained as latent episomes in the tissues of MCF-developing also validated our tiling array design in the detection of viral gene calves and rabbits, a signature of classical γ-herpesvirus latency. expression during AlHV-1 infection. Our hypothesis suggesting that MCF might be a consequence of latency was further confirmed by the high frequency of infected Cellular and Viral Gene Expression Profiles During MCF. We experi- CD8+ T cells in the cellular infiltrates of MCF-developing ani- mentally infected calves with AlHV-1 to induce MCF. All AlHV-1– mals. Persistence in actively dividing cells during γ-herpesvirus infected calves developed typical prostration, nasal discharge, lymph- latency is dependent on the expression of the latency-associated adenopathy, and severe persistent hyperthermia at 12.2 ± 0.5 d p.i. nuclear antigen (LANA) homolog, also termed genome mainte- (SI Appendix,Fig.S1A). All AlHV-1–infected animals developed nance protein, encoded by ORF73 (18). Thus, we addressed the typical MCF with lymph node (LN) hypertrophy, increased CD8+ role of AlHV-1 latency in MCF by producing ORF73-deficient T-cell percentages in peripheral blood and LN, increased IFN-γ viruses. Whereas ORF73 disruption did not impair viral replica- production, and characteristic infiltrations of lymphoblastoid cells in tion in vitro and in vivo at the first days postinfection (p.i.), a lack the perivascular spaces of many tissues (SI Appendix,Fig.S1). of ORF73 rendered AlHV-1 unable to induce MCF and persist in To determine cellular and viral gene expression during MCF, we vivo. Of importance, we finally showed that infection with an extracted RNA from the inguinal LN of each calf for analysis on ORF73-deleted recombinant virus conferred protection against a custom-designed array as described above. The choice of inguinal a lethal challenge with a WT virus. Altogether, our findings suggest LN as the selected tissue was arbitrary and based on the fact that that AlHV-1 ORF73-mediated latency is a prerequisite for the AlHV-1 viral genomic load is the highest in peripheral LN. Cellular induction of the lymphoproliferative lesions observed in MCF and and viral RNA transcription profiles were analyzed identically to bring prospects on future vaccine development for populations viral gene expression during lytic infection in vitro (two-color dye- living in areas where AlHV-1 infection is endemic. swap analyses of four independent biological repeats). We observed in the LN of MCF-developing calves a total of 225 Results − differentially expressed (P value ≤ 10 4, fold change was >4or<−4) Design of a Combined Array to Detect Viral and Cellular Gene genes, from which 55 were overexpressed and 170 were underex- Expression. We designed an oligonucleotide microarray combin- pressed during MCF (SI Appendix, Fig. S4 and Table S2). Among ing 17,408 tiling 60-mer probes complementary to each strand of the highest overexpressed genes, we detected the cytotoxic granule the AlHV-1 genome and 43,603 probes of the Bovine Gene Ex- proteases granulysin and granzymes, proinflammatory secreted pression Microarray (Agilent Technologies). In this design, more proteins [including IFN-γ,TNF-α, chemokine (C-C motif) ligand than 70% of the array consisted in cellular probes. After having fi (CCL)-3, CCL-4, and chemokine (C-X-C motif) ligand (CXCL)- veri ed that more than 75% of the overall probes were not dif- 11], and proteins involved in cell division and proliferation (in- ferentially expressed, a classical norm exp background correction cluding DNA helicase Pif1, cyclin A2 and B1, CDC28 protein ki- followed by loess normalization was applied. To visualize the viral nase regulatory subunit 2, and cell division cycle associated 2). tiling array data, we averaged the log ratio fluorescence intensities 2 Among underexpressed proteins, we found many receptors and for every probe overlapping each nucleotide in the AlHV-1 ge- transmembrane proteins, proteins involved in cellular adhesion, nome. To analyze differentially expressed cellular probes, a linear and three genes that have been involved in tumor suppression model with a dye-swap blocking variable was fit to the data using (Dab2, Armcx1, and Reck). Overall, these data show a severe the least squares method. An empirical Bayes-moderated t statistic disruption of host gene expression in LN tissue during MCF. was then used to test the AlHV-1/Mock contrast, and a Benjamini– We next analyzed the AlHV-1 genome tiling array to determine Hochberg multiple testing correction was applied. the viral gene expression in LN during MCF. The mean log2 ratio B Detection of Viral Gene Expression During Lytic Infection. We first intensities per nucleotide are shown in Fig. 1 . The results of each SI Appendix examined the expression of viral polyadenylated RNA from individual repeat in ,Fig.S3show that the analyses infected [multiplicity of infection (moi) = 0.01] madin-darby bo- were highly reproducible, with nearly all nucleotide positions vine kidney (MDBK) cells at 72 h p.i.. This late time point was showing very little variation between the four biological replicates. A chosen, because we expect the majority of viral genes to be Compared with productive infection (Fig. 1 ), viral RNA expres- expressed. The expression of viral RNA at each nucleotide position sion during MCF was very sparse throughout the AlHV-1 genome, obtained from two-color dye-swap analyses of four independent with only 10% of each strand being expressed. The expression biological repeats is shown in Fig. 1A compared with the annotated levels of some transcripts (i.e., ORF52, ER13, and ORF73) were ORF of the AlHV-1 genome. Overall, RNA expression was ob- similar to the signal intensities observed during productive in- served on 45% of the total length of both strands of the AlHV-1 fection, suggesting that the observed scarce viral gene expression is genome. The results of each individual repeat are plotted in SI not caused by low sensitivity. We detected the expression of only Appendix,Fig.S2and show very little signal variation, indicating 16 annotated ORFs (ORF17, -23, -45, -46, -A6, -52, -53, -55, -58, high reproducibility. The characteristic progressive loss of signal -59, -60, -61, -62, -65, -69, and -73) and 8 ERs that were not pre- from the 3′ to the 5′ end of most transcripts can be explained by the dicted as an ORF (ER1, -11, -12, -13, -14, -15, -16, and -19) (Fig. 1B use of oligo-dT primers in the amplification step before hybrid- and SI Appendix,TableS1). Apart from the high expression of ization. Expressed viral transcripts were, therefore, identified ORF73, which encodes the LANA homolog or genome mainte- using the wavelet transform as a multiscale peak detector on the nance protein (18), the role in γ-herpesvirus infection of most of averaged viral signal of each strand. Briefly, peaks with a nearby these genes remains largely unknown. Moreover, the expression polyadenylation signal were defined as 3′ ends, and corresponding levels of genes known to be essential and/or highly expressed 5′ ends were defined manually. Most signal peaks corresponded during productive infection, such as ORF8 (gB), ORF9 (DNA to previously annotated ORF boundaries, which indicated that polymerase), ORF22 (gH), ORF25 and -26 (capsid proteins), and the designed tiling array detected known expressed transcripts. ORF50 (reactivation transactivator), were under the detection

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Fig. 1. Genome-wide viral RNA expression during AlHV-1 infection in vitro and during MCF. (A) MDBK cells were infected with AlHV-1 at moi = 0.01, and total RNA was extracted 72 h p.i., labeled, and hybridized to the custom-designed array (n = 4). (B) Inguinal LNs were harvested from mock- or AlHV-1– infected calves developing MCF (n = 4). Total RNA was extracted, labeled, and hybridized to the array. The mean fluorescence ratio of Cy5 and Cy3 dyes

between AlHV-1– and mock-infected samples of probes overlapping each nucleotide position is plotted on a log2 scale for each strand. The red and blue curves show the forward and reverse strands of the genome, respectively. Red and blue block arrows represent annotated genes on the forward and reverse strands of the genome, respectively. Peaks identified by the wavelet transform close to a poly-A signal (AATAAA or ATTAAA) are indicated with dotted lines. Expressed regions are shown with gray boxes (dark gray, peak detection with poly-A signal and overlapping with a predicted ORF; medium gray, peak detection with poly-A signal but no overlapping with a predicted ORF; light gray, peak detection with no poly-A signal). E, expressed region not overlapping an annotated ORF.

level. These observations, therefore, strongly suggest that AlHV-1 Infiltration of ORF73-Expressing T Cells in MCF Lesions. Latent in- infection is not productive during MCF. fection of proliferating cells by γ-herpesviruses is associated with the expression of the genome maintenance protein encoded by Episomal Maintenance of AlHV-1 Genomes During MCF. Whereas ORF73 in AlHV-1 (20). To examine ORF73 expression in lesions, linear viral genomes are found in cells supporting a productive in- we produced antibodies specific to AlHV-1 ORF73 C-terminal fection, herpesvirus latency is characterized by the maintenance of domain by DNA immunization (SI Appendix, SI Materials and covalently closed viral episomes (18). We, therefore, used Gardella Methods). Specific intranuclear staining was verified after tran- gel electrophoresis to analyze the virus genome conformation in sient cell transfection with p73-V5 and on infected cells in vitro SI Appendix A B MCF. AlHV-1 genomes were almost exclusively circular in PBMCs ( ,Fig.S5 and ). Tissue cryosections were obtained and LNs of MCF-developing calves and rabbits, respectively (Fig. from MCF-developing calves or rabbits after AlHV-1 infection. fi 2A). Episomal AlHV-1 genomes were also predominant in PBMCs, Staining speci city was controlled using tissue from mock-infected LNs, spleen cells, and leukocytic cells from liver and kidney of animals or naive mouse serum as primary antibody on tissue from MCF-developing animals (Fig. 3 A, Left and B, Left). Specific MCF-developing rabbits (Fig. 2 A and B). Serial dilutions of BAC intranuclear staining was only observed in MCF-developing ani- plasmid DNA and rabbit LN cells followed by Gardella gel elec- ∼ – × 5 5 mal tissues using anti-73C polyserum. We observed high expres- trophoresis gave an estimation of 2 5 10 episomes per 10 cells sion of ORF73 in LNs of calves and rabbits (Fig. 3 A and B C (Fig. 2 ). This calculation was based on the similar signal intensities and SI Appendix,Fig.S5C and D). The experimental rabbit × 6 observed for 1.6 and 8 ng BAC plasmid DNA and 0.45 and 0.9 10 model was then used to investigate ORF73 expression in cells 6 cells, respectively (1 ng 150 kbp AlHV-1 genome ≅ 6.2 × 10 cop- infiltrating the tissues. We observed that a high number of in- ies). A similar analysis performed on bovine lymphocytic cell lines filtrating T cells expressed the ORF73 protein (Fig. 3 B–E). (LCLs) propagated from MCF-developing calves also showed epi- The percentages of ORF73-expressing cells in the cellular in- somal persistence in these lines (19). These results support classical filtrates were estimated to be around 20% in liver and lung γ-herpesvirus latency during MCF. infiltrates (Fig. 3E). Some infected cells might express low levels of

Palmeira et al. PNAS Early Edition | 3of10 Downloaded by guest on September 28, 2021 We then used the rabbit model to induce MCF. After infection, the development of MCF was monitored at regular intervals with particular attention to body temperature and hypertrophy of the popliteal LN. Rabbits infected with the 73null or 73ns recombinant strain did not develop MCF-typical hyperthermia or lesions and survived the infection (Fig. 5 and SI Appendix, Fig. S13 A–C), whereas control groups developed hyperthermia, LN and spleen hypertrophy, and MCF-typical histopathological lesions. CD8+ T-cell expansion and proliferation were further monitored by flow cytometry analyses and in vivo BrdU incorporation. Rabbits infected with the 73null or 73ns recombinant strain showed no CD8+ T-cell expansion or proliferation (SI Appendix, Figs. S7 A–C and S13D). Together, these results show that the lack of ORF73 renders AlHV-1 unable to induce MCF.

AlHV-1 Persistence in Vivo Depends on ORF73 Expression. ORF73 orthologs have been shown to be critical for maintenance of the viral genome in latently infected cells (18). To measure the viral load in vivo after rabbit infection, viral genome copy numbers were determined in lymphoid tissue by quantitative PCR (qPCR) Fig. 2. Episomal maintenance of AlHV-1 genomes during MCF. (A) Gardella during MCF (Fig. 6 and SI Appendix, Fig. S13E). Although AlHV- gel analysis of AlHV-1 genome conformation during MCF in calf peripheral 1 viral load increased exponentially in PBMCs a few days before blood or rabbit popliteal LN cells (lanes 1 and 2 show the results obtained death in rabbits infected with the WT and revertant strains, no from two different animals). Cells were lysed in situ, and extracts were virus could be detected after infection with the 73null strain at any electrophoresed through a 0.8% agarose-Tris-Borate EDTA gel before A – fi time point after infection (Fig. 6 ). At time of euthanasia, com- Southern blotting performed with AlHV-1 speci c probes. BAC plasmid DNA parable virus reactivation was observed in cocultures of LN cells was used as positive control to detect covalently closed circular (CCC) viral DNA. AlHV-1–infected MDBK cells supporting a lytic replication [±90% cy- isolated from rabbits infected with WT or 73-Rev strains, but no topathic effect (CPE)] were used to detect linear virus genome. (B) Analysis infectious center could be detected in LN cells from rabbits null of AlHV-1 genome conformation during MCF in rabbit lymphoid and non- infected with the 73 strain (Fig. 6B). This result was not caused lymphoid tissues as in A.(C) Gardella gel analysis of MCF-developing rabbit by a reactivation defect, because no viral DNA was detected after popliteal LNs. Fivefold dilutions of BAC plasmid DNA control and twofold infection with the 73null or 73ns strains in PBMCs, LNs, and spleen decreasing cell numbers are shown. cells; also, the expected viral loads were observed in animals infected with the control strains (Fig. 6C and SI Appendix, Fig. S13E). These results show that the deletion of AlHV-1 ORF73 ORF73 that could be undetectable by immunostaining. There- fore, a single-cell RT-PCR approach was conducted on cloned impairs viral persistence in MCF-susceptible rabbits. rabbit LN CD8+ T cells from mock-infected or MCF-developing Antiviral Humoral Response in Absence of ORF73. Although the lack rabbits (SI Appendix,Fig.S6). This approach enables amplifi- of ORF73 expression renders AlHV-1 unable to persist in vivo, the cation of both viral ORF73 cDNA and viral genomic DNA to 73null strain should induce an antiviral response. Thus, we exam- increase the chances of detection. We detected as much as 43% ined the antiviral antibody response after infection with the 73null (10/23) to 78% (18/23) of infected CD8+ T cells in the pop- strain. Specificanti–AlHV-1 antibodies could be detected as soon liteal LN. Together with previous observations, these results – strongly suggest that the lesions occurring during MCF can as 6 10 d after infection and reached a peak of production just fi before death in rabbits infected with the WT or 73-Rev strain (SI be attributed to the proliferation and in ltration of latently Appendix null infected T cells. , Fig. S8). Animals infected with the 73 virus also de- veloped an anti–AlHV-1 antibody response. This response was, AlHV-1 ORF73 Is Not Essential for Viral Replication but Is Essential for however, reduced at the late time points p.i. compared with the WT the Induction of MCF. The role of AlHV-1 latency in the patho- and 73-Rev groups. These results show that the deletion of ORF73 genesis of MCF was subsequently addressed by producing does not impair the development of antiviral antibody response. null ns ORF73-deleted (73 ) and -nonsense (73 ) recombinant viruses null together with their respective revertant strains (Fig. 4 and SI Infectionwith73 VirusProtectsAgainstaChallengewith Appendix Pathogenic WT AlHV-1. Induction of an antiviral response after in- , Fig. S9). The lack of ORF73 did not affect protein null expression levels of the envelope gp115 complex in infected cells fection with the apathogenic 73 strain could protect against an- SI Appendix A B SI Ap- other challenge with the pathogenic WT strain. To test this ( , Fig. S10 and ) or viral growth in vitro ( null pendix, Fig. S10C). Absence of virus growth defect in culture was hypothesis, we infected rabbits i.v. with the 73 strain before in line with some reports showing that γ-herpesvirus ORF73 is not a challenge 28 d later with the WT virus by intranasal inoculation to essential for virus replication in vitro (21–24), although MuHV-4 mimic the natural infection route (Fig. 7A). Control groups received or Rhesus monkey rhadinovirus showed lytic replication deficit a mock-infected inoculum. The development of MCF was moni- under certain conditions or enhancement, respectively (25, 26). tored daily for hyperthermia and hypertrophy of popliteal LNs. All null To further address the effect of ORF73 deletion on virus repli- rabbits preinfected with the 73 strain survived the challenge, cation in vivo, we used the luciferase+ WT AlHV-1 (247N-luc+) whereas four of five mock-infected rabbits developed MCF after the BAC plasmid (15) to produce a 247N-luc+-73null recombinant challenge (Fig. 7B). Antibody ELISA showed an effective anti– virus (SI Appendix, Fig. S11). We then infected rabbits intranasally AlHV-1 antibody production in rabbits preinfected with the 73null and monitored luciferase signals by ex vivo bioluminescence im- strain (Fig. 7C). Flow cytometry analyses of PBMCs revealed that aging in the nasal turbinates and the lungs (SI Appendix, Fig. S12). rabbits preinfected with the 73null strain did not show CD8+ T-cell We observed that disruption of ORF73 did not impair host colo- proliferation, whereas all rabbits developing MCF in the control nization at days 2 and 4 p.i., suggesting that ORF73 is not essential group had highly increased CD8+ T-cell percentages (Fig. 7D). for virus replication in vivo. Finally, rabbits showing protection against challenge did not

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Fig. 3. In situ expression of AlHV-1 ORF73 during MCF. (A) Immunostaining of ORF73 expression in cryosections of calf axillary LNs and rabbit popli- teal LNs in mock-infected animals (mock) or dur- ing MCF (AlHV-1). White arrows show positive cells with intranuclear ORF73 staining. (B)Immu- nostaining of ORF73 expression in rabbit popliteal LNs, spleen, lung, liver, and kidney tissue during MCF. Microphotographs are centered on infil- trative lesions. (Magnification: 200×.) (C)Spleen cryosection showing intranuclear ORF73 staining. White arrows show positive cells with intranuclear ORF73 staining. (D) ORF73 and CD3 costaining of rabbit spleen and kidney cryosections. Immunos- MICROBIOLOGY tainings were performed with homemade anti- 73C mouse polyserum. Stainings were revealed with Alexa-Fluor 488 nm goat anti-mouse IgG secondary antibody (AF488) and DAPI used for counterstaining. (Magnification: 100×.) (E)Per- centage of ORF73-positive cells in lung (n =10) and liver (n = 12) cellular infiltrates of two MCF- developing rabbits. Infiltrative areas were pho- tographed and delimited before the number of DAPI+ nuclei was counted using ImageJ software and the cell counter plugin followed by the de- termination of ORF73+ cell numbers.

develop typical perivascular infiltration of lymphoblastoid cells in to explain how AlHV-1 infection leads to disease development in the tissues as opposed to the unprotected control group (Fig. 7E). MCF-susceptible species. Our recent observations in the rabbit model suggested that AlHV-1 might be predominantly not pro- Discussion ductive during MCF. In this study, we first sought to extend our Herpesviruses coevolve with their natural host species, which findings obtained with the experimental rabbit model to species often results in the virus adaptation to its host. AlHV-1 is a good developing MCF in natural conditions. We, therefore, studied example of coevolution, where the virus is so adapted to its MCF experimental induction after AlHV-1 infection of calves. natural host that it is able to persist without causing any clinical We designed a high-density tiling array of the entire AlHV-1 sign or lesion, resulting in the infection of virtually the entire genome to examine polyadenylated viral RNA expression in vitro wildebeest population. Contrastingly, AlHV-1 transmission to and during MCF in calves. This kind of approach has been used other ruminant species (e.g., cattle) causes the development of before in various herpesvirus infections (27–29), and a recent MCF that, ultimately, leads to the death of the affected animal. report on MuHV-4 infection has further shown the robustness of This relationship between AlHV-1 and wildebeest could be seen whole-genome tiling array analyses (29). Of the 90 genomic as only beneficial for the virus. However, there is also the pos- regions expressed during productive infection in cell culture, our sibility that this relationship results from an evolutionary sym- analyses identified only 16 annotated ORFs and 8 unpredicted biotic adaptation for both species. Under this hypothesis, AlHV-1 regions that were transcribed in LNs during MCF. Interestingly, would take advantage of its adaptation to its host to persist, and nearly all annotated ORFs were expressed during productive in- wildebeests would benefit from MCF induction in competing fection in vitro, and we detected the expression of 23 additional species, because it would provide access to more food and/or ERs that were not assigned to an ORF. Non-ORF ERs were also weakened sick animals for large predators during the wildebeest detected in other γ-herpesvirus infection (29, 30), which redefines calving period, corresponding to the period when virus trans- the genetics of viral RNA expression during AlHV-1 infection and mission mostly occurs. Understanding how AlHV-1 is able to the need of future experiments to address the role of these ad- induce MCF to susceptible species like cattle is an important step ditional ERs during AlHV-1 infection. Consistent with our pre- to the understanding of this unique evolutionary relationship vious observations, ORF73 was expressed in MCF, whereas no between a virus and its hosts. expression of genes essential for virus productive infection was Despite various theories, mostly limited to the description of detected. In addition, we observed the expression of additional the characteristic infiltrative lesions, no clear view currently exists viral genes with mostly unknown function in infection (Fig. 1 and

Palmeira et al. PNAS Early Edition | 5of10 Downloaded by guest on September 28, 2021 Fig. 4. Generation of the ORF73-deleted (73null) virus strain. (A) Recombineering methodology used to delete the entire ORF73 coding sequence by galK insertion and generation of the 73null and 73-Rev strains. (B) Flowchart of stages performed to produce the 73null and 73-Rev strains. (C) Southern blotting analysis of produced BAC plasmids and reconstituted strain genomic DNA after HindIII restriction. The probes used for Southern blotting are indicated: C500, entire BAC WT plasmid DNA digested with SacI; ORF73, PCR amplicon corresponding to nucleotides 116,438–116,681 of the AlHV-1 genome; galK, entire galK coding sequence. (D) Confocal microscopy of MDBK infected with the BAC WT, 73null, and 73-Rev strains and stained with anti-73C polyserum as primary antibody and Alexa 568 nm-conjugated goat anti-mouse IgG (AF568) as secondary antibody. Nuclei were counterstained with TO-PRO-3 iodide (TOPRO3).

SI Appendix, Tables S1 and S4). It remains to be determined antibodies, showing that a high frequency of T cells infiltrating whether these genes and ERs are important in the pathogenesis. the tissues of lymphoid and nonlymphoid organs expressed the la- Interestingly, a recent report on OvHV-2–caused MCF in cattle tency-associated protein (Fig. 3). Moreover, we also showed using has identified the sole expression of ORF73 and an unpredicted a single-cell RT-PCR approach that a very high percentage of region corresponding to AlHV-1 ER16 (31). Although no func- CD8+ T cells (43–78%) (SI Appendix,Fig.S6) in the LNs of MCF- tion could be attributed to this region, it might be involved in the developing rabbits is infected by AlHV-1. Based on these results pathogenesis of MCF. and together with previous data, we can conclude that latency is Our previous results in the rabbit model showed the low or most probably a prerequisite for the induction of MCF. This hy- absence of productive virus infection in MCF (14, 15), and we pothesis is consistent with latency-mediated malignancies observed confirmed and extended these results in a whole-genome approach in some human γ-herpesvirus infections, such as EBV or Kaposi in calves. However, it remained unclear whether AlHV-1 persis- sarcoma-associated herpesvirus (KSHV) (32, 33). tence was mediated by classical episomal maintenance in pro- To determine the role of AlHV-1 latency-associated ORF73 in liferating cells or rather, explained, for instance, by a succession of MCF, we used the 73null and 73ns recombinant viruses, both im- abortive lytic cycles. We observed by Gardella gel electrophoresis paired for the expression of ORF73. Growth kinetics analyses in performed directly on lymphocytic cells isolated from MCF- permissive bovine nasal fibroblasts and testis primary cell cultures developing calves and rabbits that AlHV-1 genomes were essen- did not reveal any significant deficit of the AlHV-1 ORF73- tially circular in both species and also in cells infiltrating non- deficient viruses compared with WT and the revertant strains, lymphoid tissues such as liver and kidney, strongly suggesting suggesting that ORF73 is not necessary for virus replication. γHV- classical γ-herpesvirus latency in MCF with episomal persistence 68 ORF73-deficient virus displayed substantial growth deficit in of AlHV-1 genomes. This result is important, because it further murine fibroblasts at very low moi (25), and ORF73 disruption in validates the rabbit experimental model and also supports the Rhesus monkey rhadinovirus generated a highly lytic virus strain hypothesis of a proliferation of latently infected cells being re- (26). Other than these two examples, most of the ORF73-deficient sponsible for MCF (14, 15). A causative role of latently infected γ-herpesviruses tested so far have not shown any growth defect in proliferating CD8+ T cells implies that a high percentage of T cells vitro (21–24, 34). These reports suggest that ORF73 might affect infiltrating the perivascular spaces in MCF are infected. Infected viral growth in cell culture only under certain specific conditions. cells in situ were detected using homemade anti-ORF73 polyclonal Very few in vivo animal models are readily available to test viral

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Fig. 5. ORF73 deletion renders AlHV-1 unable to induce MCF. (A) Body temperature was recorded daily after rabbit infection with the WT, 73null, or 73-Rev recombinant viruses. Mock-infected rabbits received uninfected BT cells. Rabbits were identified according to the day of euthanasia p.i. (see numbers). (B) Cumulative incidence of survival of the four groups of four rabbits infected i.v. with mock-infected BT cells or BT cells infected with the WT, 73null, or 73-Rev virus strains. (C) Spleen and popliteal LN weight at the time of euthanasia. Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA with Bonferroni posttest). (D) Histopathological characterization of MCF lesions observed in kidney, liver, and lung of one rabbit representative of each group. White arrowheads indicate typical infiltrations of lymphoblastoid cells. A, arteriole; Bi, small bile duct; Br, bronchi; Hp, hepatocyte; RC, renal cor- puscule; T, uriniferous tubule; V, vein. Equivalent results were obtained in two independent experiments.

replication at the primary site of infection in vivo. Intranasal in- velop typical perivascular infiltrations of lymphoblastoid cells in fection of mice with MuHV-4 ORF73-deficient viruses revealed kidney, liver, or lung. Moreover, we did not observe any modifi- similar lung infectious titers (22) or attenuation in the acute lung cation of the percentages of proliferating CD8+ cells using in vivo infection (23, 34). In an attempt to clarify whether viral replication BrdU incorporation after infection with the ORF73-deficient occurs at the primary site of infection in absence of ORF73 ex- strains, whereas CD8+ cells severely proliferated after infection pression, we produced and used a 247Nluc+-73null recombinant with the WT or revertant strains as observed before (14). The five virus to infect rabbits intranasally. This approach enabled the de- strains used in this study were produced with a limited number of tection of virus host colonization as soon as 2 d p.i. in the lung and passages in cell culture (less than five) and showed unaltered the nasal turbinates (SI Appendix,Fig.S12). Ex vivo biolumines- molecular genomic structure and unaffected replication capability. cence analyses showed that the disruption of ORF73 in AlHV-1 High passage in cell culture has, indeed, been shown to produce did not impair host colonization at the early time points p.i. and apathogenic AlHV-1 strains (36–38). There is, however, no evi- even led to increased signal intensities in the nose and lungs of dence of any implication of ORF73 in the loss of virulence because some rabbits. Infection with a MuHV-4 ORF73-deficient virus high passage. The causative role of ORF73 in MCF was observed resulted in replication that was concentrated to the inoculum site, in independent experiments using normalized infectious doses suggesting that MuHV-4 ORF73 could be important for effective between each strain and two independently engineered mutant host colonization (35). To date, host colonization of AlHV-1 after strains. Also, some of the rabbits were kept up to 6 mo after primary infection is poorly understood, and future experiments are infection and did not show any MCF clinical sign or lesion, needed to elucidate how AlHV-1 invades MCF-susceptible hosts. further supporting that the deletion of ORF73 renders AlHV-1 Nonetheless, we provide in this study good indications that a lack of apathogenic. ORF73 does not affect viral replication in vivo, at least up to 4 d p.i. The genome maintenance protein encoded by ORF73 in Monitoring of MCF induction in rabbits after infection with γ-herpesvirus tethers the viral episome to cellular chromosomes 73null and 73ns recombinant viruses showed that a lack of ORF73 during cell division and therefore, is essential for virus persistence expression in AlHV-1 infection of rabbits resulted in their survival, in vivo (22–24). One of the theories to explain MCF induction whereas all rabbits infected with WT or revertant strains suc- was that AlHV-1 infection could create an autoimmune-like en- cumbed to the infection with typical clinical signs and lesions. vironment, resulting in the trans activation and proliferation of Rabbits infected with 73null and 73ns virus strains did not show uninfected T cells. After activation, these uninfected cells would hyperthermia or hypertrophy of spleen and LNs and did not de- not need any more presence of virus to amplify and cause MCF.

Palmeira et al. PNAS Early Edition | 7of10 Downloaded by guest on September 28, 2021 transcriptomic interactions and/or immune evasion properties that might be involved in MCF pathogenesis. In this study, we provide strong evidence indicating that latency is at the heart of MCF. The mechanisms behind the onset of the pathology, however, are not fully understood but most likely, do not involve overabundance of lytic replication and consequent inflammation. Our bioluminescence analyses using 247N-luc+ WT and 73null viruses suggested that ORF73 is not necessary for viral replication in vivo after primary infection and that AlHV-1 ini- tially replicates in nasal turbinates and lungs. From then, we can hypothesize that, after entering the organism and infecting CD8+ T cells caused by AlHV-1 lymphotropism, the virus establishes latency in these cells. Although it is possible that the virus could only be a passenger in CD8+ T cells and trigger proliferation in- dependently of latency-dependent mechanisms, our findings rather support that the deregulation and proliferation of CD8+ T cells have a direct link with latent infection. Indeed, we showed abundant AlHV-1 genomes that are essentially circular during MCF, identified high frequencies of infected CD8+ Tcells,and showed that disruption of ORF73 rendered AlHV-1 apathogenic, whereas it did not significantly affect initial virus replication in vivo. According to this hypothesis, we can expect that latently infected CD8+ T cells present at the peak of the disease would Fig. 6. Loss of viral persistence in absence of ORF73. (A) Viral load was assayed by qPCR of viral genome copies in PBMCs over time after infection of result from the clonal expansion of only few mother cells infected rabbits with the WT, 73null, or 73-Rev recombinant strains. Real-time PCR after host colonization. Future experiments addressing this point quantification was normalized on β-globin cellular genomic sequence. Data need to be developed. For instance, it would be interesting to + are plotted as individual measurements (n = 4). (B) Infectivity from popliteal determine the T-cell receptor polymorphism of infected CD8 T LN cells was tested by infectious center assay of single-cell suspensions on cells and whether only infected CD8+ T cells are proliferating in MDBK-overlaid media containing 0.6% carboxymethylcellulose to obtain MCF. The exact mechanisms used by AlHV-1 to trigger pro- isolated syncytia as described in SI Appendix, SI Materials and Methods. Data liferation remain to be deciphered. Although ORF73 could be are plotted as means ± SEM (n = 4). (C) qPCR of viral genome copies in null directly involved in tumorogenesis, such as observed in KSHV popliteal LN at the time of euthanasia of rabbits infected with the WT, 73 , malignancies (32), AlHV-1 also encodes numerous genes and or 73-Rev recombinant strains. Real-time PCR quantification was normalized on β-globin cellular genomic sequence. Data are plotted as means ± SD of predicted microRNAs of yet unknown functions that could be triplicate measurements for each sample (n = 4). Equivalent results were involved in processes involving, for example, cell cycle and/or obtained in two independent experiments. apoptosis deregulation, mechanisms that are directly involved in malignancies induced by other γ-herpesvirus infection (32, 45–47). The identification of viral genes and regions expressed in MCF in However, MCF induction is associated with an exponential in- our tiling array opens a perspective for future identification of such creasing of viral genomes at the later time points p.i., whereas no mechanisms. Additionally, microarray analyses of host gene dif- virus genome copy could be detected in PBMCs or lymphoid ferential expression identified numerous cellular genes that could tissue after infection with the apathogenic ORF73-deficient virus be involved in the pathogenesis of MCF. Although the possible strains. Given that ORF73 is not essential for viral replication in interpretations of these data are substantially limited, because it is vitro or in vivo at early time points p.i., we can speculate that not possible to distinguish a direct effect of AlHV-1 infection on ORF73-mediated AlHV-1 episomal maintenance in vivo repre- gene expression from an indirect effect because of the disruption sents an essential prerequisite for the development of MCF. As of the tissue itself (i.e., the difference in the cellular population a consequence, proliferation of CD8+ T cells in MCF would be phenotypes and proportions in Mock and MCF samples), we can caused by cis-acting mechanisms. Whether ORF73 is solely nec- conclude that MCF is associated with a strong disruption of the essary for virus persistence in vivo or has additional pathogenic LN gene expression profile with up-regulation of proinflammatory function to induce the observed acute lymphoproliferative lesions cytokines, cytotoxic proteins, and genes involved in cell division. is unknown and will need additional investigations. Although the Additional investigations on sorted cell subsets, such as CD8+ T main function of KSHV ORF73 is to maintain the viral episome cells, should identify differential expression of genes directly (39), it has been shown that LANA can also directly interfere with caused by AlHV-1 infection and determine the involvement of important antitumorigenic pathways, such as, for examples, the such an inflammatory environment on the development of MCF inhibition of p53-mediated functions (40, 41), RB–E2F tumor lesions. Indeed, it has been shown in virus-induced malignancies, suppressor pathways (42), and antiproliferative TGF-β signaling such as Kaposi sarcoma, that host cytokines and growth factors are (43). KSHV LANA is also able to up-regulate survivin expression necessary to ensure optimal tumorogenesis (32). and promote B-lymphoma cell proliferation (44). AlHV-1 encodes AlHV-1 infection of MCF-susceptible species is problematic the largest genome maintenance protein described to date (24). It in endemic regions and also zoos, where high value and endan- is, therefore, possible that AlHV-1 ORF73 might similarly have gered susceptible species are kept alongside wildebeest. Vacci- prooncogenic functions that could ultimately be involved in the nation attempts have been performed with variable success (48–50). triggering of CD8+ T-cell proliferation observed in MCF. It is However, all strategies reside on the use of attenuated or inacti- unknown at this stage which ORF73 domains are involved in the vated WT virus, and full protection was unfortunately never ob- genome maintenance function. However, the C-terminal domain tained. We took advantage of the apathogenic 73null virus to induce shows similarities with the ones from other γ-herpesviruses, which aprotectiveresponseintherabbitmodel.Weobtainedastrong is indicative of conserved genomic episome maintenance functions antibody response and full protectionafteranintranasalchallenge (18). The large internal acidic repeat domains that are conserved with a pathogenic WT virus. ORF73-deficient γ-herpesviruses have not at the sequence level but in regards to their structural prop- previously been shown to elicit effective protection against WT erties are suggestive of conserved functions, such as cellular challenge (22, 34, 51) and have the unique advantage of being

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Fig. 7. 73null Infection induces protection against MCF after challenge with WT AlHV-1. (A) Experimental procedure. Three groups were used, each consisting of five rabbits. The 73null/WT group received 105 pfu 73null strain i.v., whereas the two remaining mock/WT and mock/mock groups received uninfected vehicle BT cells. At 28 d p.i., rabbits of the 73null/WT and mock/WT groups were challenged intranasally with 105 pfu WT pathogenic strain. The mock/mock group received PBS intranasally. (B) Cumulative incidence of survival of the rabbits of each group. (C) Antibody indirect ELISA for detection of anti–AlHV-1 anti- bodies in serum of rabbits throughout the experiment. (D) Percentages of CD8+ cells in the gated T-cell population analyzed by flow cytometry at regular intervals throughout the experiment. (E) Histopathological characterization of MCF lesions observed in kidney, liver, and lung of one rabbit representative of each group. White arrowheads indicate typical infiltrations of lymphoblastoid cells. Av, arteriole; Bi, small bile duct; Hp, hepatocyte; RC, renal corpuscule; T, uriniferous tubule; V, vein. Equivalent results were obtained in two independent experiments.

unable to persist. Although the specific protection mechanisms Technologies). Additional procedures are given in SI Appendix, SI Materials remain unknown, this recombinant AlHV-1 strain could induce and Methods. a protective response in MCF-susceptible species and represent fi a serious candidate vaccine. Availability of such vaccine will bring Viral Transcript Identi cation. The delimitations of viral transcripts were prospects for effective protection against MCF for the local pas- inferred from the mean base pair log2 fold change signal in the in vitro experiment (Fig. 1A). Because of the poly-dT amplification step, transcripts toralist population living in regions where cattle and wildebeest showed a characteristic shape defined by a sharp peak on their 3′ end. These herds coexist. peaks were identified using the wavelet transform as a multiscale shape fi Altogether, the ndings detailed in this study suggest that detector in LastWave (http://www.cmap.polytechnique.fr/~bacry/LastWave). AlHV-1 infection of MCF-susceptible species induces a periph- We used the first derivative of the Gaussian as the analyzing wavelet and eral T-cell lymphoma-like pathology, where ORF73 expression determined the positions of the peaks as the abscissa to which the signal and latency are both essential. maxima converged. For each abscissa, we retained signal peaks (i) bearing a canonical polyadenylation signal within ±100 bp (AATAAA or ATTAAA)

Materials and Methods and (ii)withlog2 fold change that was above a 0.6 threshold. These peaks fi ′ ′ Animals and Virus Infection in Vivo. Eight healthy 4- to 6-mo-old Holstein– were used to de ne the 3 ends of transcripts. Corresponding 5 ends were fi Friesan calves, free from Bovine herpesvirus 1 and 4 and Bovine Viral Diarrhea de ned manually from the remaining peaks and by checking the local signal virus, were nurtured at the Veterinary and Agrochemical Research Center shape. A few transcripts were manually added in regions that had failed to (Brussels, Belgium). Specific pathogen-free NZW rabbits were purchased from be detected by our wavelet-based method. The overall expression of tran- the Centre d’économie rurale (Marloie, Belgium) and used at 8–12 wk of age. scripts was computed as the mean log2 fold change along their length. Animals were inoculated i.v. with ∼3 × 106 mock or AlHV-1–infected bovine Transcripts with overall expression that was above a 0.4 threshold were turbinate (BT) cells. AlHV-1–infected cells were harvested when CPE reached discussed in the text. ≥90% and normalized using ORF3 qPCR (SI Appendix, SI Materials and Methods). Animals were examined daily for clinical signs and rectal tempera- Production of AlHV-1 BAC Recombinant Plasmids and Viruses. The AlHV-1 BAC ture. According to bioethical rules, calves and rabbits were euthanized after clone was used to produce the recombinant plasmids using the galactokinase 48 h of persistent hyperthermia (>40 °C). The local ethics committees of the (galK) gene as selection marker in Escherichia coli (52). Additional proce- Veterinary and Agrochemical Research Center and the University of Liège for dures are given in SI Appendix, SI Materials and Methods. experiments involving calves and rabbits, respectively, have accredited the performed animal studies. Statistical Analysis. Microarray analyses and figures were conducted using R and the limma package from the Bioconductor project (53–55) combined Microarray Design and Hybridization. A custom microarray combining a high- with ad hoc programs written in Python. All other statistical analyses were density tiling array of the entire AlHV-1 genome with a set of probes tar- conducted using GraphPad Prism 4 software. geting all bovine transcripts was designed for 8 × 60,000 glass slides (Agilent Additional procedures are given in SI Appendix, SI Materials and Methods.

Palmeira et al. PNAS Early Edition | 9of10 Downloaded by guest on September 28, 2021 ACKNOWLEDGMENTS. The authors thank Dr. H. Li and Prof. D. M. Haig for calves as well as J. Piret and F. Massart for samples processing. L.P. is a providing the antigen-coated ELISA plates and the AlHV-1 C500 virus strain, Postdoctoral Fellow from the University of Liège. O.S. is a Research Fellow of respectively. We thank Dr. F. Ectors for providing assistance in micromanip- the Fonds pour la formation à la Recherche dans l’Industrie et dans l’Agri- ulation of single cells. For technical assistance, we thank the personnel of the culture (FRIA). F.M. and B.G.D. are a Research Fellow and a Research Asso- Veterinary and Agrochemical Research Center for the in vivo experiments in ciate of the Fonds de la Recherche Scientifique, respectively.

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