Claire A M Becker

Laurence Malandrin

Thibaut T Larcher

Alain Chauvin

Emmanuel Bischoff

Sarah I Bonnet
email: sbonnet@vet-alfort.fr

Validation of BdCCp2 as a marker for Babesia divergens sexual stages in ticks

Keywords:

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Babesiosis is a worldwide disease due to intraerythrocytic protozoan parasites from the genus Babesia. It is known as a source of emerging zoonosis and its public health impact on livestock and humans is ongoing [START_REF] Genchi | Human babesiosis, an emerging zoonosis[END_REF][START_REF] Gray | Zoonotic babesiosis: overview of the disease and novel aspects of pathogen identity[END_REF][START_REF] Vannier | Update on babesiosis[END_REF]. The disease causes principally fever and anemia but also hemoglobinuria, hyperbilirubinuria, and possibly organ failure [START_REF] Homer | Babesiosis[END_REF]. Babesia parasites are transmitted to the mammalian host by ticks. In Europe, Babesia divergens is the most frequent species infecting domestic cattle [START_REF] Zintl | Babesia divergens, a bovine blood parasite of veterinary and zoonotic importance[END_REF] and is transmitted by the hard tick Ixodes ricinus. This bovine parasite is also thought to be responsible for most European cases of human babesiosis [START_REF] Häselbarth | First case of human babesiosis in Germany -Clinical presentation and molecular characterisation of the pathogen[END_REF][START_REF] Martinot | Babesiosis in immunocompetent patients, Europe[END_REF]. Some cases of human babesiosis attributed to other Babesia species, namely Babesia sp. EU1 [START_REF] Häselbarth | First case of human babesiosis in Germany -Clinical presentation and molecular characterisation of the pathogen[END_REF][START_REF] Herwaldt | Molecular characterization of a non-Babesia divergens organism causing zoonotic babesiosis in Europe[END_REF] and Babesia microti [START_REF] Hildebrandt | First confirmed autochthonous case of human Babesia microti infection in Europe[END_REF], have been recently reported.

Very few data are available concerning the life cycle of Babesia and its sexual reproduction inside the tick vector. In fact, only asexual reproduction within the salivary glands of vector ticks and within the blood cell of vertebrates have been identified with certainty [START_REF] Mehlhorn | The piroplasms: life cycle and sexual stages[END_REF]. When a tick feeds on an infected vertebrate, where the parasite multiplies by binary asexual division in red blood cells, it can acquire some piroplasms in its blood meal. Inside the tick, only parasite sexual stages, the gametocytes, survive and evolve to different steps of development: gamogony and sporogonies in various tick organs [START_REF] Mehlhorn | The piroplasms: life cycle and sexual stages[END_REF]. The first step of this development is the sexual reproduction inside the tick gut, which involves male and female gametocytes. Gametocytes were discovered for Babesia bigemina by Koch in 1906 and were called Strahlenkörper (ray bodies) [START_REF] Koch | Beiträge zur Entwicklungsgeschichte der Piroplasmen[END_REF]. After that, they were described for different Babesia species inside ticks [START_REF] Friedhoff | Rediscovery of Koch's ''Strahlenkörper'' of Babesia bigemina[END_REF][START_REF] Karakashian | Ultrastructural studies on sporogony of Babesia microti in salivary gland cells of the tick Ixodes dammini[END_REF][START_REF] Mehlhorn | Light and electron microscopic study on developmental stages of Babesia canis within the gut of the tick Dermacentor reticulatus[END_REF]. More recently B. bigemina sexual stages could be in vitro produced in red blood cells without tick antigen and were isolated without the use of specific markers [START_REF] Mosqueda | Babesia bigemina sexual stages are induced in vitro and are specifically recognized by antibodies in the midgut of infected Boophilus microplus ticks[END_REF][START_REF] Vichido | Expression analysis of heat shock protein 20 and rhoptry-associated protein 1a in sexual stages and kinetes of Babesia bigemina[END_REF]. Concerning B. divergens, only two studies report the visualization of potential sexual stages inside the tick gut [START_REF] Bonnet | Transstadial and transovarial persistence of Babesia divergens DNA in Ixodes ricinus ticks fed on infected blood in a new skin-feeding technique[END_REF][START_REF] Mackenstedt | Sexual cycle of Babesia divergens confirmed by DNA measurements[END_REF]. Some markers permitting the identification of gametocytes with certainty are needed to refine knowledge on such stages and moreover to investigate the mechanisms that control parasite transmission as well as the relationships between parasites and tick vectors.

We previously identified three B. divergens genes ( bdccp1, bdccp2 and bdccp3) thanks to a post-genomic bioinformatics analysis based on sequence identity between the available genomes of Plasmodium spp. and Babesia spp. [START_REF] Becker | Identification of three CCp genes in Babesia divergens: novel markers for sexual stages parasites[END_REF]. The 3 genes are orthologs of Plasmodium genes that are sexual stage specific and belong to the well-conserved Apicomplexan CCp (Limulus Coagulation factor C domain proteins) family. Antibodies were produced against one of these proteins, BdCCp2, to further study protein expression. Tested on Plasmodium berghei gametocytes, the results showed that these antibodies specifically recognized hemoprotozoan gametocytes and not parasite asexual stages. In the present study, these antibodies were used to validate their ability to specifically recognize tick-specific parasite stages among which are necessarily the sexual stages of B. divergens and subsequently, the capacity of BdCCp2 to be used as a marker of B. divergens gametocytes.

Material and methods

2.1. Parasite culture B. divergens (Bob2A clone) was cultured in bovine red blood cells in Roswell Park Memorial Institute 1640 medium (Lonza, Basel, Switzerland) and 20% Fetal Calf Serum (Lonza, Basel, Switzerland) as previously described [START_REF] Chauvin | Sheep as a new experimental host for Babesia divergens[END_REF], until 10% parasitemia was attained. The bovine blood used in all experiments was obtained from parasite-free cows (tested by culture) reared at the Ecole Nationale Vétérinaire de Nantes.

Ticks

Parasite-free I. ricinus females, reared in the laboratory, were artificially engorged and infected with a parasite culture using an in vitro feeding system as previously described [START_REF] Bonnet | Transstadial and transovarial persistence of Babesia divergens DNA in Ixodes ricinus ticks fed on infected blood in a new skin-feeding technique[END_REF], and used for immunohistochemistry analysis. Ticks engorged with non-infected blood were used as negative controls. In addition, partially engorged ticks were collected on cows in a farm where the presence of B. divergens had been previously attested (56% of cows positive by IFA-Immunofluorescence Antibody Assay) (La Verrie, France) [START_REF] Becker | Natural transmission of zoonotic Babesia spp. by Ixodes ricinus ticks[END_REF]. The serological status of the cows was evaluated and, on the whole sampling, 17 ticks collected on 9 seropositive cows and 17 others collected on 9 seronegative cows were studied (Table 1).

BdCCp2 recombinant polypeptides synthesis and antisera production

Two different antisera, directed against the same BdCCp2 region, were produced in parallel as previously described and were validated as able to recognize P. berghei gametocytes [START_REF] Becker | Identification of three CCp genes in Babesia divergens: novel markers for sexual stages parasites[END_REF].

Briefly, a recombinant polypeptide of 284 amino acids named rpBdCCp2 and based on the BdCCp2 sequence was produced in bacteria, and used to generate a specific immune serum (R serum) by subcutaneous immunization of a rabbit (Charles River Laboratories, L'Arbresle, France). A 15 amino acids synthetic peptide, included in the rpBdCCp2 sequence (Eurogentec, Seraing, Belgium) designated spBdCCp2, and based on the P. falciparum PfCCp2 immunogenic peptide [START_REF] Pradel | A multidomain adhesion protein family expressed in Plasmodium falciparum is essential for transmission to the mosquito[END_REF][START_REF] Pradel | Plasmodium falciparum: codependent expression and co-localization of the PfCCp multi-adhesion domain proteins[END_REF], was also used to generate guinea pig antiserum (G serum) by the Eurogentec company (''Super speedy 28 day polyclonal antibody production''). Here both sera were used for the detection of sexual stages of B. divergens both by immunoblot and immunohistochemistry analysis.

Immunohistochemistry analysis

Five partially engorged female ticks, 3 experimentally infected by membrane feeding technique and 2 non-infected negative controls, were fixed, in their entirety, for 48 h in 10% neutral buffered formalin and embedded in paraffin wax. For immunohistochemical analyses, 4 lm thick sections were cut, dewaxed and pretreated for 6 min with trypsin (1 mg/ml) (Sigma) at 37 °C and in 3% hydrogen peroxide (Gifrer, Decines, France) for 10 min at room temperature. Sections were then blocked for 20 min with 20% normal goat serum (Dako, Glostrup, Denmark).

R and G sera, diluted at 1:500 and 1:10 respectively, were used as primary antibodies and incubated on slides in 2% Bovine Serum Albumin (Sigma) for 1 h at 37 °C. Corresponding pre-immune sera were used as negative controls. The slides were then incubated with anti-rabbit (Dako) or anti-guinea pig (Jackson Immunoresearch Laboratories) biotinylated secondary antibodies in 2% Bovine Serum Albumin for 30 min and revealed with streptavidin (Dako) and DAB Liquid Substrate for immunoperoxidase (Dako), according to the manufacturer's instructions. The slides were counterstained with Gill's hematoxylin (Surgipath, Peterborough, UK) and examined under a microscope.

SDS-PAGE and immunoblot analysis

Immune sera were tested on in vitro cultures of B. divergens parasites and on gut contents of ticks collected on cows. The thirtyfour field-collected ticks and one non-infected control tick were dissected in a small drop of PBS (Phosphate Buffered Saline) and the tick guts were frozen at À20 °C until use. Protease Inhibitor Cocktail (Sigma) was added to all samples (3 ll for 250 ll of sample) just before freezing.

Individual tick guts and parasite culture samples (250 ll) were washed with 1 ml of PBS, lysed with 1 ml of 0.01% saponin (Sigma) at room temperature, and washed 3 times with 1 ml of PBS. Pellets were treated for 5 min at 100 °C with SDS-PAGE Sample Buffer (v/ v) (Tris-HCl 62.5 mmol/l, Glycerol 10%, ß-mercaptoethanol 2%, Bromophenol Blue 0.1%, SDS 2%), centrifuged for 30 s and 20 ll of the collected supernatant was analyzed on a 8% acrylamide SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis) gel under reducing conditions. The proteins were then transferred (3 h at 50 mA) onto a nitrocellulose membrane (Bio-Rad) with a TG powder buffer (Interchim, Montluçon, France). After a saturation step of 30 min with Tris buffered saline and 0.1% Tween-20 (Sigma) (TBSt) containing 10% non-fat milk, the membrane was incubated with G serum (1:250) for 30 min in TBSt milk 5%. After washing, the membranes were incubated for 30 min with Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Guinea Pig IgG (Jackson ImmunoResearch Laboratories, Newmarket, UK), diluted 1:10,000 in TBSt milk 5% and developed in a solution of 5-Bromo-4-chloro-3-indolyl phosphate and nitro-blue tetrazolium chloride (BCIP Ò /NBT, Sigma).

Ethics statement

This study was carried out in strict accordance with the good animal care practice of the recommendations of the European guidelines. The protocol was approved by the Committee the Ethics of Animal Experiments of the national Veterinary School of Nantes and all efforts were made to minimize suffering of animals.

Results

Detection of B. divergens sexual stages by immunohistochemistry on artificially infected ticks

Immunohistochemical analyses were performed with R and G sera on partially engorged ticks that were experimentally infected. The BdCCp2 protein was detected in piriform or ovoid bodies, measuring 2.5-4.5 lm in diameter, in the tick gut (Fig. 1C andD). Similar labeled elements, comparable with previously described sexual stages of Babesia spp., were obtained with both sera (the R serum giving a better signal). Isolated bodies or sometimes clusters of two bodies were scattered in the blood meal. Asexual stages, although largely present in the blood meal (as attested by Giemsa staining of the adjacent serial section of the sample, Fig 1B ), were not recognized by the sera. Roughly a dozen labeled organisms were observed in a coronal section of experimentally infected ticks: this low proportion, in relation to the parasite population as a whole, could be consistent with gametocyte frequency. No labeling of the blood meal was obtained for non-infected engorged ticks (Fig. 1E andF) or with non-immune sera on infected engorged tick (Fig. 1G andH).

Detection of BdCCp2 by immunoblot analysis in B. divergens infected erythrocytes and in naturally infected ticks

Protein samples from the digestive tract of partially engorged ticks collected on cows and from an in vitro culture of B. divergens in bovine erythrocytes were run on SDS-PAGE and challenged with G serum by Western blot. Red Ponceau coloration performed before the immunostaining showed that a similar amount of proteins was loaded in each lane (data not shown). Results obtained for the 34 tested ticks are presented in Table 1. The BdCCp2 protein was detected in 8 naturally infected ticks out of the 34 tested, as a discrete band of approximately 171 kDa, whereas no signal was detected in the laboratory-reared non-infected tick (Fig. 2A). It is noticeable that BdCCp2 was only detected inside ticks that engorged on seropositive cows and that among those ticks, signal was not systematically detected (Table 1). Furthermore, when ticks were collected on the same cow, some of them gave positive result and other a negative one in Western blot (data not shown). Finally, the BdCCp2 protein was not detected in protein samples from red blood cells infected by B. divergens (Fig. 2B).

Discussion

Babesiosis has been recently considered emerging zoonosis and Babesia spp. correspond to the second most common blood-borne parasites of mammals after the trypanosomes [START_REF] Häselbarth | First case of human babesiosis in Germany -Clinical presentation and molecular characterisation of the pathogen[END_REF]. Nevertheless, a lot of data are missing concerning the life cycle of these parasites, in particular their acquisition by and their development inside the tick vector. To understand conditions of transmission from vertebrate host to vector, one necessary point is to study and determine the conditions of the gametocytes appearance and the factors that rule their acquisition by the tick. The characterization of B. divergens sexual stages using molecular tools permitting accurate identification of gametocytes is hence essential to study parasite biology and monitor transmission. With this aim in view, we used an in silico post-genomic approach in a previous work, to identify genes that are sexual stage specific in B. divergens [START_REF] Becker | Identification of three CCp genes in Babesia divergens: novel markers for sexual stages parasites[END_REF]. Three genes bdccp1, bdccp2 and bdccp3 were identified and the mRNA of these 3 genes were detected in the intraerythrocytic parasite population [START_REF] Becker | Identification of three CCp genes in Babesia divergens: novel markers for sexual stages parasites[END_REF]. Such results suggested that the B. divergens population inside the vertebrate host is already committed to the sexual pathway, as reported for Plasmodium genus [START_REF] Lavazec | Analysis of mutant Plasmodium berghei parasites lacking expression of multiple PbCCp genes[END_REF][START_REF] Pradel | A multidomain adhesion protein family expressed in Plasmodium falciparum is essential for transmission to the mosquito[END_REF]. Bdccp genes, specific of gametocytes, could then be used as molecular markers, through the detection of their mRNA, to evaluate the production of sexual stages in the vertebrate host [START_REF] Becker | Identification of three CCp genes in Babesia divergens: novel markers for sexual stages parasites[END_REF]. We previously produced antisera directed against a recombinant peptide of one of the identified proteins, BdCCp2. As the gametocytes of B. divergens have not be isolated to date, the absence of possible positive control forced us to validate this protein as a marker of hemoprotozoan gametocytes, since the antisera recognized the intraerythrocytic sexual stages of P. berghei [START_REF] Becker | Identification of three CCp genes in Babesia divergens: novel markers for sexual stages parasites[END_REF]. In the present work our aim was to evaluate the specificity of these antisera for B. divergens gametocytes and thus, the possibility to use BdCCp2 as a marker for parasite sexual stages.

Immunohistochemistry analysis revealed that a few parasites, similar in size to the sexual stages of other Babesia spp. [START_REF] Friedhoff | Rediscovery of Koch's ''Strahlenkörper'' of Babesia bigemina[END_REF][START_REF] Mehlhorn | Light and electron microscopic study on developmental stages of Babesia canis within the gut of the tick Dermacentor reticulatus[END_REF][START_REF] Mosqueda | Babesia bigemina sexual stages are induced in vitro and are specifically recognized by antibodies in the midgut of infected Boophilus microplus ticks[END_REF][START_REF] Rudzinska | Intraerythrocytic 'gametocytes' of Babesia microti and their maturation in ticks[END_REF], were marked by anti-BdCCp2 sera, within the tick gut. These labeled elements confirm the existence of B. divergens sexual stages inside the tick as already suggested both by DNA measurements [START_REF] Mackenstedt | Sexual cycle of Babesia divergens confirmed by DNA measurements[END_REF] and microscopical observations [START_REF] Bonnet | Transstadial and transovarial persistence of Babesia divergens DNA in Ixodes ricinus ticks fed on infected blood in a new skin-feeding technique[END_REF]). Yet it is currently impossible to determine whether these marked parasites are B. divergens gametocytes just after their arrival in the tick, gametes just before fusion, or zygotes. IFA, another method that should permit to observe parasites with a finer resolution, was tested but a too high background due to tick gut content prevented us to visualize parasites (data not shown). Thus, our results confirm that the BdCCp2 protein is expressed by a minute part of B. divergens parasites inside the tick and can be used there as a marker of B. divergens sexual stages.

The protein BdCCp2 was not detected in intraerythrocytic stages of B. divergens, neither by Western blot nor by IFA (data not shown) on infected bovine erythrocytes, showing that, in vitro, the protein was not expressed in the B. divergens asexual population. In contrast, BdCCp2 was detected inside B. divergens infected ticks, either artificially from in vitro culture or naturally engorged. Western blot analysis showed, in naturally engorged ticks, a band at 171 kDa, the molecular weight estimated for BdCCp2 [START_REF] Becker | Identification of three CCp genes in Babesia divergens: novel markers for sexual stages parasites[END_REF]. Low signal strength was probably due to the very small amount of BdCCp2 protein that is present inside whole tick gut protein extract, sexual stages being only a minute part of the parasite population. In addition to get a signal on the blot, we had to incubate nitrocellulose membrane with the substrate for about 15 min, explaining the high level of background. Nonetheless, our assay revealed the presence of BdCCp2 in only 8 out of the 34 ticks tested, and only among the 17 ticks that were collected on seropositive cows. That could be explained by the fact that except at the onset of infection, seronegative cows usually do not carry B. divergens, leading in most cases to parasite-free engorged ticks. In contrast, not all the ticks that fed on seropositive cows showed BdCCp2 signal. Even if a previous study indicated a good correlation (89.6%) between a positive serological status and intraerythrocytic asymptomatic carriage detected by in vitro culture [START_REF] Malandrin | Isolation of Babesia divergens from carrier cattle blood using in vitro culture[END_REF], the frequency of gametocytes should be even lower compared to that of asexual stages and these forms may not be present in the blood meal ingested by the tick. It is noticeable that two ticks collected on the same seropositive cow did not show the same result. The presence of negative and positive ticks collected from the same cow indicates either that transmission to the tick is not always effective or that the low amount of transmitted gametocytes is not systematically detectable with our method. Moreover some cows fed ticks that were all detected positive whereas others cows only gave negative ticks. It could have been interesting to test the production of sexual stages by searching the presence of bdccp2 mRNA in cow blood.

Finally there is apparently a discrepancy between the times of transcription and translation of this gene. As we could show in a previous publication [START_REF] Becker | Identification of three CCp genes in Babesia divergens: novel markers for sexual stages parasites[END_REF] bdccp2 is transcribed but not translated to a protein in the vertebrate host. In the present work we could detect the protein only inside the vector. This could be explained by the fact that the parasite population might be already committed to the sexual pathway inside the vertebrate host but might only express sexual stage proteins under specific conditions that are only encountered inside the vector. It is then concomitant with the morphological transformation observed only once in the tick for other Babesia species [START_REF] Mehlhorn | The piroplasms: life cycle and sexual stages[END_REF][START_REF] Smith | Sexual differentiation and sex determination in the Apicomplexa[END_REF]. The functions of B. divergens BdCCp2 protein need to be further studied and established. As BdCCp2 contains adhesive domains, like all the members of the apicomplexan CCp family [START_REF] Becker | Identification of three CCp genes in Babesia divergens: novel markers for sexual stages parasites[END_REF][START_REF] Dessens | LCCL proteins of apicomplexan parasites[END_REF], it might be involved in specific events happening only inside the tick, such as adhesion of gametes, as it is reported for Plasmodium spp. [START_REF] Kuehn | Family members stick together: multi-protein complexes of malaria parasites[END_REF][START_REF] Simon | Sexual stage adhesion proteins form multi-protein complexes in the malaria parasite Plasmodium falciparum[END_REF].

Conclusion

We confirmed the existence of B. divergens sexual stages inside the vector tick I. ricinus. We also showed that transmission of B. divergens from cow to tick cannot be considered fully efficient at the tick population level, even if all required conditions are present. Our investigations permitted development of tools that will be very useful to study B. divergens biology. Thanks to our molecular markers, methods could be developed to quantitatively monitor the sexual stages of this parasite inside the vertebrate host and to follow the parasites inside the vector. These tools may be used in epidemiological studies in order to characterize and quantify parasite transmission. Understanding the parameters that govern the transmission of Babesia from the vertebrate host to the tick, should help explain the appearance of the clinical cases of babesiosis and pave the way to novel approaches of disease control.

A B

Fig. 1 .

 1 Fig. 1. Coronal sections artificially partially engorged female Ixodes ricinus ticks analyzed by immunohistochemistry. An adult tick, partially engorged and experimentally infected with clone Bob2A of Babesia divergens (A, B, G, and H), was tested with R serum (rabbit serum directed against a recombinant polypeptide of BdCCp2) to detect BdCCp2 protein. (A) May Grunwald Giemsa stain of B. divergens in whole tick section; cuticle (Cut) and blood meal (BM) are indicated. (B) High magnification of the blood meal (BM); some colored asexual parasites present in erythrocytes are indicated with arrows. (C) and (D) Immunochemistry detection of sexual parasites with high magnification of the blood meal composed of hemacytic debris; round or piriform labeled elements of 2.5-4.5 lm (arrow) were scattered in the blood meal. (E) and (F) Control non-infected tick challenged with R serum. (G) and (H) Experimentally infected tick challenged with pre-immune serum. (E) and (G) Blood meal is delineated by enterocytes of the digestive endothelium (DE). Enzymes contained in the cytoplasm of the enterocytes are responsible for an unspecific signal as assessed by the observation that a similar signal was present on experimentally infected tick challenged without primary and secondary antibodies.

Fig. 2 .

 2 Fig. 2. BdCCp2 detection by Immunoblot analysis of gut contents of partially engorged I. ricinus fed on B. divergens seropositive cows (A) and red blood cell populations (B). Protein extracts were challenged with G serum (guinea pig serum directed against a synthetic peptide of BdCCp2, spBdCCp2). (A) Lanes 1-7: gut content extract of some ticks engorged on serologically positive cows. Ticks 1-5 give a positive result when tick 6 and 7 give a negative one. Lane C: gut content extract from a negative control tick. The band corresponding to BdCCp2 (around 171 kDa) is indicated by an arrow. Double band around 200 kDa corresponds to non-specific reactions against red blood cell proteins. (B) Lane 1: non-infected bovine erythrocytes; Lane 2: B. divergens (clone Bob2A) in vitro infected bovine erythrocytes. No positive signal is observed.

Table 1

 1 BdCCp2 protein detection by Western blot in I. ricinus ticks collected on cows from a B. divergens infected cow herd. The serological status of cows was evaluated by Immunofluorescence Antibody Assay against B. divergens antigens.

	Cow serological status	Western blot result (number of ticks)
	(number of cows)
	Total (34)	Protein detected (8)	No signal (26)
	+(17)	8	9
	À(17)	0	17

Acknowledgments

We are grateful to Maggy Jouglin (INRA), Agnès Bouju-Albert (ENVN), Béatrice Oberhauser (ENVN) and Lydie Guigand (ENVN) for technical assistance, to Peter H. David (Institut Pasteur) for helpful discussions and English corrections and to the ''Tiques et Maladies à Tiques'' group (REID-Réseau Ecologie des Interactions Durables) for stimulating discussions. This work is part of CB's PhD thesis, which was funded by INRA and Region Pays de Loire. EB was funded by the Délégation Générale pour l'Armement (DGA n 22120/DSP/SREAF and n 04 34 025).


Fabienne Archer
email: fabienne.archer@univ-lyon1.fr

Jean-François Mornex

Caroline Leroux

Interplay Between JSRV, an Oncogenic Retrovirus, and the Pulmonary Epithelium

Keywords: Lung, JSRV, immune response, adenocarcinoma, alveolar type II cells

Jaagsiekte sheep retrovirus (JSRV) is a retrovirus which infects small ruminants and is responsible for a natural lung cancer. This virus bears an oncogenic envelope inducing epithelial cell transformation from the deep lung. The cells, main target of the infection and constitutive of the tumors, are alveolar type II cells in the alveoli and Clara cells in the bronchioli. The immune response to JSRV infection is poorly understood. The specific humoral response is limited and the cell-mediated response is marked by CD4+ lymphocytopenia, neutrophilia, and macrophage invasion of the tumor. The mechanisms of viral immune evasion could be explained by the direct and indirect immunosuppressive effects of the virus, and by the immune tolerance of the infected hosts due to the presence in the sheep genome of endogenous viral forms (enJSRV).

sentinel and epithelial cells, are able to detect intracellular dsRNA (double strand RNA) and ssRNA (single strand RNA) to initiate type I IFN and pro-inflammatory cytokine production via activation of specific-transcription factors including IRFs (Interferon Regulatory Factors ) and NF-B (Nuclear Factor-kappaB).

In recent years, it has become clear that airway epithelial cells not only constitute a mechanical barrier, but also actively contribute to the innate immune system [START_REF] Bals | Innate immunity in the lung: how epithelial cells fight against respiratory pathogens[END_REF]. Although there have been numerous studies on viral infection of the conducting airways (reviewed in [START_REF] Johnston | Overview of virus-induced airway disease[END_REF]) relatively few have focused on the distal gas-exchange unit of the deep lung, site of severe clinical infection such as SARS-CoV (severe acute respiratory syndrome-coronavirus) or avian influenza virus. The epithelium where gas exchanges occur is constituted of two main cell types. Alveolar type I cells are flat terminally-differentiated cells that cover about 95% of the alveolar surface area and are mainly involved in gas exchange. Cuboidal alveolar type II cells are metabolically active, produce pulmonary surfactant and transport fluid and sodium to the basolateral surface to keep the alveolar surface suitable for gas exchange [START_REF] Mason | Biology of alveolar type II cells[END_REF]. Alveolar type II cells are self-renewing and are able to transdifferentiate into alveolar type I cells when they need to be replaced. Both alveolar type I and type II cells participate in the innate immune response by producing interferons and cytokines in response to virus infection [START_REF] See | Innate immune response to viral infection of the lungs[END_REF][START_REF] Miura | Rat coronaviruses infect rat alveolar type I epithelial cells and induce expression of CXC chemokines[END_REF][START_REF] Wang | Differentiated human alveolar type II cells secrete antiviral IL-29 (IFN-lambda 1) in response to influenza A infection[END_REF]. They also produce antimicrobial substances such as surfactant protein A (SP-A), surfactant protein-D (SP-D), beta-defensins or lysozyme in response to microbial infection [START_REF] Bals | Innate immunity in the lung: how epithelial cells fight against respiratory pathogens[END_REF][START_REF] Crouch | Surfactant protein-D and pulmonary host defense[END_REF][START_REF] Doss | Interactions of alpha-, beta-, and theta-defensins with influenza A virus and surfactant protein D[END_REF]. SP-A and SP-D interact with the glycoconjugates and lipids on the virus surface inducing aggregation of complexes, enhancement of phagocytosis and release of cytokines and chemokines at the site of infection [START_REF] Kishore | Surfactant proteins SP-A and SP-D: structure, function and receptors[END_REF]. Beta-defensins have a dual role in antiviral activity : they may block viral infection by directly acting on virions or they may affect the target cell and thereby indirectly interfere with viral infection [START_REF] Klotman | Defensins in innate antiviral immunity[END_REF]. Taken together, all these mechanisms take place in order to eliminate viruses from the lung and maintain the integrity of the alveolar function. Some viruses have developed strategies to bypass or delude this defence in order to initiate cell deregulation or even tumours.

OVINE PULMONARY ADENOCARCINOMA: A RETROVIRUS-INDUCED LUNG CANCER

JSRV (Jaagsiekte Sheep RetroVirus) is a -retrovirus, infecting small ruminants. This virus is responsible for a pulmonary adenocarcinoma associated with the transformation of epithelial cells from the lung parenchyma, i.e. alveolar type II cells in the alveoli and Clara cells in the bronchioli [START_REF] Griffiths | Pathology and pathogenesis of ovine pulmonary adenocarcinoma[END_REF] (Fig. 1). The disease is usually sporadic within flocks but can be epizootic in some specific conditions of farming, leading to the death of up to 5% of the animals [START_REF] Griffiths | Pathology and pathogenesis of ovine pulmonary adenocarcinoma[END_REF]. This animal tumour is clinically, radiologically and histologically related to the pneumonic form of human bronchioloalveolar cancer (pBAC), a rare form of lung tumour in humans which represent less than 4% of all adenocarcinomas [START_REF] Griffiths | Pathology and pathogenesis of ovine pulmonary adenocarcinoma[END_REF][START_REF] Read | The epidemiology of bronchioloalveolar carcinoma over the past two decades: analysis of the SEER database[END_REF]. These similarities have always intrigued pathologists and chest physicians and have been stressed as early as 1939 [START_REF] Mornex | Pathology of human bronchioloalveolar carcinoma and its relationship to the ovine disease[END_REF][START_REF] Bonne | Morphological resemblance of pulmonary adenomatosis (jaaksiekte) in sheep and certain cases of cancer of the lung in man[END_REF]. Both the pneumonic form of human lung adenocarcinoma and the ovine pulmonary adenocarcinoma are described as mixed-type adenocarcinoma, associating a predominance of bronchioloalveolar lesions and papillary and/or acinar lesions. Ovine pulmonary adenocarcinoma has been considered as a model for human adenocarcinomas, and especially for the pneumonic-type bronchioloalveolar cancer [START_REF] Mornex | Pathology of human bronchioloalveolar carcinoma and its relationship to the ovine disease[END_REF]. Similarly to ovine pulmonary adenocarcinoma, bronchioloalveolar cancer is a slow-growing tumour with rare distant metastases. It is clinically associated with highly productive cough and progressive restrictive respiratory failure [START_REF] Mornex | Pathology of human bronchioloalveolar carcinoma and its relationship to the ovine disease[END_REF][START_REF] Wislez | Bronchioloalveolar carcinoma[END_REF]. The disease is usually sporadic within flocks but can be epizootic in some specific conditions of farming, leading to the death of up to 5% of the animals [START_REF] Griffiths | Pathology and pathogenesis of ovine pulmonary adenocarcinoma[END_REF].

Given the similarities between ovine pulmonary adenocarcinoma and human BAC, a viral cause to human bronchioloalveolar cancer has long been hypothesized; but up to now reports exploring the link between JSRV and BAC [START_REF] De Las Heras | Evidence for a protein related immunologically to the jaagsiekte sheep retrovirus in some human lung tumours[END_REF][START_REF] Hiatt | Lack of DNA evidence for jaagsiekte sheep retrovirus in human bronchioloalveolar carcinoma[END_REF][START_REF] Yousem | Absence of jaagsiekte sheep retrovirus DNA and RNA in bronchioloalveolar and conventional human pulmonary adenocarcinoma by PCR and RT-PCR analysis[END_REF] remain controversial. Nevertheless, ovine pulmonary adenocarcinoma offers an invaluable animal model for studying the molecular mechanisms of lung epithelial transformation occurring in cancer.

JSRV AND ITS ONCOGENIC ENVELOPE

JSRV genome contains the typical retrovirus genes gag, pro, pol and env respectively encoding the capsid and nucleocapsid proteins, the protease, the enzymatic activities and the envelope glycoproteins. An additional small ORF (open reading frame), called "x" and encoding a putative protein of unknown function, overlaps the end of the pol gene (Fig. 2). JSRV mainly infects and only transforms alveolar type II cells and Clara cells [START_REF] Platt | Alveolar type II cells expressing jaagsiekte sheep retrovirus capsid protein and surfactant proteins are the predominant neoplastic cell type in ovine pulmonary adenocarcinoma[END_REF] leading to development of tumours in the deep lung. Using molecular detection of RNA and DNA, JSRV has been demonstrated to infect not only the respiratory tract but also several lymphoid tissues such as the mediastinal lymph nodes draining the lungs [START_REF] Palmarini | Jaagsiekte retrovirus establishes a disseminated infection of the lymphoid tissues of sheep affected by pulmonary adenomatosis[END_REF]. JSRV has been detected at low level in the spleen, the thymus, the bone marrow and the peripheral blood mononuclear cells [START_REF] Palmarini | Jaagsiekte retrovirus establishes a disseminated infection of the lymphoid tissues of sheep affected by pulmonary adenomatosis[END_REF]. Although very few cells of the immune systems are infected, the highest provirus load is in the monocytes/macrophages, followed by B and T cells. There is no evidence of virus transformation of any of these cell types [START_REF] Palmarini | Jaagsiekte retrovirus establishes a disseminated infection of the lymphoid tissues of sheep affected by pulmonary adenomatosis[END_REF][START_REF] Holland | Jaagsiekte retrovirus is widely distributed both in T and B lymphocytes and in mononuclear phagocytes of sheep with naturally and experimentally acquired pulmonary adenomatosis[END_REF]. Hyal2 (Hyaluronidase type 2) is the cell receptor for JSRV [START_REF] Rai | Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation[END_REF] and is expressed at the surface of many cell types. Hyal2 expression is not restricted to lung epithelial cells, suggesting that the entry of the virus into the cells is not the sole determinant of pathogenesis.

A family of related endogenous sequences, enJSRV closely related to the exogenous JSRV, is present in domestic and wild ungulates [START_REF] Hecht | Distribution of endogenous type B and type D sheep retrovirus sequences in ungulates and other mammals[END_REF]. JSRV and enJSRV genomes are highly related with 90-98% homology in deduced amino-acid sequences [START_REF] Palmarini | Molecular cloning and functional analysis of three type D endogenous retroviruses of sheep reveal a different cell tropism from that of the highly related exogenous jaagsiekte sheep retrovirus[END_REF]. Endogenous JSRVs derive from ancestral integration of exogenous JSRV into the host genome. They are maintained as Mendelian genes and are vertically transferred to the offspring. They are defective for at least one viral gene as a result of mutations acquired during their integration into the germ-line. The involvement of enJSRV in the carcinogenesis remains unknown.

It is now clearly established that JSRV induces tumors via the oncogenic properties of its envelope, Env, which is both necessary and sufficient to induce transformation (reviewed in [START_REF] Leroux | Jaagsiekte Sheep Retrovirus (JSRV): from virus to lung cancer in sheep[END_REF]). The oncogenic property of JSRV Env has been shown both in vitro in various cell types including murine NIH 3T3 fibroblasts [START_REF] Maeda | Direct transformation of rodent fibroblasts by jaagsiekte sheep retrovirus DNA[END_REF], rat 208F fibroblasts [START_REF] Rai | Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation[END_REF], avian DF-1 fibroblasts [START_REF] Allen | The jaagsiekte sheep retrovirus envelope gene induces transformation of the avian fibroblast cell line DF-1 but does not require a conserved SH2 binding domain[END_REF][START_REF] Zavala | Relevance of Akt phosphorylation in cell transformation induced by Jaagsiekte sheep retrovirus[END_REF], bronchial human BEAS-2B epithelial cells [START_REF] Danilkovitch-Miagkova | Hyaluronidase 2 negatively regulates RON receptor tyrosine kinase and mediates transformation of epithelial cells by jaagsiekte sheep retrovirus[END_REF], canine kidney MDCK epithelial cells [START_REF] Liu | Transformation of madin-darby canine kidney epithelial cells by sheep retrovirus envelope proteins[END_REF], and rat kidney RK3E cells [START_REF] Maeda | Roles of the Ras-MEK-mitogen-activated protein kinase and phosphatidylinositol 3kinase-Akt-mTOR pathways in Jaagsiekte sheep retrovirus-induced transformation of rodent fibroblast and epithelial cell lines[END_REF]; and in vivo in immunedeficient mice [START_REF] Wootton | Sheep retrovirus structural protein induces lung tumours[END_REF] and in sheep [START_REF] Caporale | Expression of the jaagsiekte sheep retrovirus envelope glycoprotein is sufficient to induce lung tumors in sheep[END_REF]. This oncogenic mechanism is unusual and JSRV is among the few retroviruses using one of its structural proteins to transform cells. Deletion experiments show that the TM (TransMembrane) region of the envelope is the main determinant for cell transformation [START_REF] Chow | Transformation of rodent fibroblasts by the jaagsiekte sheep retrovirus envelope is receptor independent and does not require the surface domain[END_REF][START_REF] Hofacre | Multiple domains of the Jaagsiekte sheep retrovirus envelope protein are required for transformation of rodent fibroblasts[END_REF][START_REF] Hull | Mutational analysis of the cytoplasmic tail of jaagsiekte sheep retrovirus envelope protein[END_REF]. The cytoplasmic tail (CT) of TM, composed of 43 amino acids, is essential for the transformation process in MDCK and NIH-3T3 cells [START_REF] Liu | Transformation of madin-darby canine kidney epithelial cells by sheep retrovirus envelope proteins[END_REF][START_REF] Palmarini | A phosphatidylinositol 3-kinase docking site in the cytoplasmic tail of the Jaagsiekte sheep retrovirus transmembrane protein is essential for envelope-induced transformation of NIH 3T3 cells[END_REF]. This region contains a peptidic YXXM motif, corresponding to a potential consensus site (phosphorylated on tyrosine Y) linked to the SH2 domain of the p85 subunit of PI3K (Phosphatidyl-Inositol-3 Kinase), a kinase that activates Akt (Fig. 3). The mechanisms leading to JSRVinduced cell transformation are in fact much more complex than previously considered. Several experiments ruled out a direct role for the YXXM motif in Akt activation [START_REF] Zavala | Relevance of Akt phosphorylation in cell transformation induced by Jaagsiekte sheep retrovirus[END_REF][START_REF] Liu | Transformation of madin-darby canine kidney epithelial cells by sheep retrovirus envelope proteins[END_REF][START_REF] Liu | Putative phosphatidylinositol 3kinase (PI3K) binding motifs in ovine betaretrovirus Env proteins are not essential for rodent fibroblast transformation and PI3K/Akt activation[END_REF].

Although a number of points remain unanswered, the transformation steps are dependent on the nature and origin of the cells. Hence, understanding the key events in the transformation of ovine alveolar type II cells is now the challenge of ongoing work. Our group has developed primary cell cultures isolated from ovine tumoral lungs [START_REF] Archer | Alveolar type II cells isolated from pulmonary adenocarcinoma: a model for JSRV expression in vitro[END_REF][START_REF] Suau | Telomerase activation in a model of lung adenocarcinoma[END_REF]; those tumour-derived cells are alveolar type II cells as revealed by the presence of lamellar bodies in the cytoplasm, and the expression of surfactant protein C (SP-C) and A (SP-A). We have reported activation of telomerase in ovine pulmonary adenocarcinoma-derived alveolar type II cells and in tumoral lung tissues, suggesting that replicative senescence may be negatively regulated in this tumour. Moreover, the Akt pathway deregulation has been shown to be deregulated in the tumoral process [START_REF] Suau | Telomerase activation in a model of lung adenocarcinoma[END_REF] (Fig. 3).

ADAPTATIVE IMMUNE RESPONSE TO JSRV

The immune response to JSRV infection is poorly understood [START_REF] Summers | Systemic immune responses following infection with Jaagsiekte sheep retrovirus and in the terminal stages of ovine pulmonary adenocarcinoma[END_REF]. The cell-mediated response is narrow with CD4+ lymphocytopenia and neutrophilia being demonstrated in the peripheral blood of adult sheep during the terminal stages of the cancer [START_REF] Holland | Jaagsiekte retrovirus is widely distributed both in T and B lymphocytes and in mononuclear phagocytes of sheep with naturally and experimentally acquired pulmonary adenomatosis[END_REF][START_REF] Rosadio | Leukocyte frequency alterations in sheep with naturally and experimentally induced lung cancer[END_REF]. More recently, a study reports invading macrophages in the tumours and its surrounding, during naturally or experimentally induced ovine pulmonary adenocarcinoma [START_REF] Summers | An influx of macrophages is the predominant local immune response in ovine pulmonary adenocarcinoma[END_REF]. Immature macrophages (CD14+, weak CD11b+) or monocytes expressing high levels of IFN-are present in the immediate vicinity of the tumour and within the alveolar lumina. Macrophage activity seems to be suppressed at the periphery of the tumour, suggesting a local immune suppressive response that may be mediated by the The gag gene encodes the matrix, the capsid and the nucleocapsid; the pro gene encodes a protease, the pol gene encodes the reverse transcriptase, RNaseH and integrase proteins; and the env gene encodes the glycoproteins needed for receptor recognition and envelope anchoring. An important feature of the retroviral genome is the long terminal repeat (LTR) regions found on both end of the provirus genome. The LTR plays an important role in initiating viral DNA synthesis and its integration as well as regulating transcription of the viral genes. The JSRV genome also contains an additional gene called "x" of unknown role.

surfactant secretion [START_REF] Summers | An influx of macrophages is the predominant local immune response in ovine pulmonary adenocarcinoma[END_REF]. No increase of dendritic, B and T cell populations has been evidenced. Older reports suggested induction of a minor immunosuppression upon JSRV infection and its association with an increased susceptibility to other infectious agents such as Pasteurella haemolityca [START_REF] Myer | The possible involvement of immunosuppression caused by a lentivirus in the aetiology of jaagsiekte and pasteurellosis in sheep[END_REF]. Reduced lymphoproliferative responses to mitogen stimulation such as concanavalin A has been shown in terminally ill adult sheep as well as in JSRV-inoculated lamb, prior to the onset of clinical disease [START_REF] Summers | Systemic immune responses following infection with Jaagsiekte sheep retrovirus and in the terminal stages of ovine pulmonary adenocarcinoma[END_REF] suggesting that JSRV may compromise the host cellular immune response.

Interestingly, DC-LAMP/CD208 (Dendritic Cell-Lysosomal Associated Membrane Protein) is strongly expressed at the apical side of alveolar type II cells in lung tissues from JSRV-infected animals and human bronchioloalveolar cancer [START_REF] Salaun | CD208/dendritic celllysosomal associated membrane protein is a marker of normal and transformed type II pneumocytes[END_REF]. DC-LAMP/CD208 has been originally described as a molecule specifically expressed in mature dendritic cells at the limiting membrane of MHC Class II-containing intracellular compartments involved in the MHC class II peptide loading and transport to the cell surface [START_REF] Barois | MHC class II compartments in human dendritic cells undergo profound structural changes upon activation[END_REF][START_REF] De Saint-Vis | A novel lysosome-associated membrane glycoprotein, DC-LAMP, induced upon DC maturation, is transiently expressed in MHC class II compartment[END_REF]. In mouse, DC-LAMP/CD208 colocalizes with MHC class II molecules in lamellar bodies, intracytoplasmic organelles specific of alveolar type II cells [START_REF] Salaun | CD208/dendritic celllysosomal associated membrane protein is a marker of normal and transformed type II pneumocytes[END_REF]. This may link DC-LAMP to the controversial role of alveolar type II cells in MHC class II restricted antigen presentation. Alveolar type II cells constitutively express MHC Class II molecules [START_REF] Cunningham | Constitutive expression of MHC and adhesion molecules by alveolar epithelial cells (type II pneumocytes) isolated from human lung and comparison with immunocytochemical findings[END_REF] increased by IFN in rats [START_REF] Schneeberger | Induction of MHC-determined antigens in the lung by interferon-gamma[END_REF] or pathological conditions in humans [START_REF] Arbustini | Coexpression of aspartic proteinases and human leukocyte antigen-DR in human transplanted lung[END_REF][START_REF] Komatsu | Phenotypic characterization of alveolar capillary endothelial cells, alveolar epithelial cells and alveolar macrophages in patients with pulmonary fibrosis, with special reference to MHC class II antigens[END_REF], cathepsin [START_REF] Guzman | ICAM-1 and integrin expression on isolated human alveolar type II pneumocytes[END_REF] or CD54 [START_REF] Guzman | ICAM-1 and integrin expression on isolated human alveolar type II pneumocytes[END_REF]. Alveolar type II cells strategically localized at the interface between the parenchyma and the outside environment, express molecules required for efficient antigen presentation to CD4+ T cells and may act as pulmonary antigen-presenting cells (APC) [START_REF] Zissel | Human alveolar epithelial cells type II are capable of regulating T-cell activity[END_REF].

Evidence of a JSRV-specific humoral response remains a subject of discussion. Some studies showed a local IgA response, the formation of viral immune complexes, and a systemic antibody response that cross-react with recombinant antigens of highly related viruses [START_REF] Verwoerd | Isolation and preliminary characterization of the jaagsiekte retrovirus (JSRV)[END_REF][START_REF] Rosati | Characterization of enzootic nasal tumor virus capsid antigen[END_REF][START_REF] Kwang | Recognition of ovine lentivirus gag gene products by serum from infected sheep[END_REF]. On the other hand, accumulating data concluded to the absence of a JSRV-specific immune response [START_REF] Sharp | Sheep pulmonary adenomatosis: demonstration of a protein which cross-reacts with the major core proteins of Mason-Pfizer monkey virus and mouse mammary tumour virus[END_REF][START_REF] Demartini | The etiology and pathogenesis of ovine pulmonary carcinoma (sheep pulmonary adenomatosis)[END_REF][START_REF] Verwoerd | Virus Infections of Ruminants[END_REF][START_REF] De Las Heras | Experimental transmission of enzootic intranasal tumors of goats[END_REF][START_REF] Ortin | Lack of a specific immune response against a recombinant capsid protein of Jaagsiekte sheep retrovirus in sheep and goats naturally affected by enzootic nasal tumour or sheep pulmonary adenomatosis[END_REF]. This failure to detect JSRV specific antibodies in experimentally or naturally infected sheep remained puzzling. But more recently, it has been shown that antibodies could be detected in the serum or lung lavages of sheep immunized with recombinant JSRV capsid or surface glycoprotein inoculated with various adjuvants [START_REF] Sharp | Natural history of JSRV in sheep[END_REF][START_REF] Summers | Jaagsiekte sheep retrovirus-specific immune responses induced by vaccination: a comparison of immunisation strategies[END_REF]. These results indicate that sheep are not inherently unresponsive to JSRV antigens and that the apparent tolerance, perhaps as a consequence of endogenous sequence expressed in utero or as a direct consequence of infection by JSRV, can be broken.

The infection of the immune system by JSRV may be advantageous to the virus either by directly facilitating the infection and the subsequent transformation of alveolar type II cells or indirectly by the induction of an immunosuppressive state. MMTV (mouse mammary tumour virus) and JSRV share interesting features. Both are retrovirus that have their own endogenous counterparts and are associated with epithelial tumours. Both viruses replicate actively in transformed epithelial cells but are also maintained at a low-level of infection in host lymphoid cells. However, the infection route and the targeted organs are distinct. MMTV enters through the digestive tract and has to reach the mammary gland, meaning that the infection of B and T cells is an absolute requirement for the virus transfer to the target organs. In the case of JSRV, initial infection is mainly through the respiratory route and therefore alveolar type II cells and Clara cells are readily accessible. It is then Works done on various cell lines have shown that the MAPK pathways can also be implicated when cells are expressing the envelope protein of JSRV [START_REF] Leroux | Jaagsiekte Sheep Retrovirus (JSRV): from virus to lung cancer in sheep[END_REF].

conceivable that JSRV first infects lung epithelial cells and replicates. After few replications, surrounding lymphoid cells or phagocytic cells may carry the virus to the mediastinal lymph nodes. Holland and colleagues have shown that dissemination to the lymphoid tissues precedes tumour formation, as detection of JSRV proviral DNA is positive as early as 7 days post-inoculation and well before the onset of tumoral lesions [START_REF] Holland | Jaagsiekte retrovirus is widely distributed both in T and B lymphocytes and in mononuclear phagocytes of sheep with naturally and experimentally acquired pulmonary adenomatosis[END_REF]. Lymphocyte proliferation studies in response to mitogen stimulation have revealed an alteration in host cellular responses due to the presence of JSRV [START_REF] Summers | Systemic immune responses following infection with Jaagsiekte sheep retrovirus and in the terminal stages of ovine pulmonary adenocarcinoma[END_REF] (reduced response to ConA stimulation). This modification occurring early in the infection course is sustained through tumorigenesis up to the terminal stage of the disease. Lastly, despite low viral load in immune cells during the early steps post infection, the presence of viruses does alter the cell functions [START_REF] Summers | Systemic immune responses following infection with Jaagsiekte sheep retrovirus and in the terminal stages of ovine pulmonary adenocarcinoma[END_REF]. This may affect the function of lymphoid tissues and interfere or prevent the development of a specific immune response against the tumoral cells or the virus allowing the tumour progression (Fig. 4). At present, the role of JSRV infection of lymphoid tissues in pathogenesis is still unknown.

The majority of cases of JSRV-induced adenocarcinomas presents with a peripheral CD4+ lymphocytopenia and neutrophilia [START_REF] Holland | Jaagsiekte retrovirus is widely distributed both in T and B lymphocytes and in mononuclear phagocytes of sheep with naturally and experimentally acquired pulmonary adenomatosis[END_REF]. Taken together with the lack of circulating JSRV antibodies and the increased susceptibility to secondary bacterial infection, this could participate to a mild immunosuppression of JSRV-infected sheep. However, it is difficult to believe that immunosuppression could explain this absence of immune response, as even the highly immunosuppressive HIV is unable to abolish the circulation of virus-specific antibodies.

enJSRV INTERFERENCE

The absence of a strong immune JSRV-specific response may be related to the presence of the closely related enJSRV sequences. During ontogeny, clonal deletion of JSRV-specific repertoire may lead to immunological tolerance. Indeed, retroelements form a large and diverse family of mobile elements that can be found in most eukaryotic organisms. They propagate by reverse transcription of RNA intermediates and integrate their genetic information into the genomic DNA of the host cells. Retroelements include retrovirus-like elements, the integrated forms or proviruses consist of two long terminal repeats (LTRs) flanking an internal region containing one to three major open reading frame coding for structural (Gag and sometimes Env) and enzymatic (Pol) functions necessary for their replication cycle. These elements, known as endogenous retroviruses (ERVs) are transmitted through the germline as stable Mendelian genes. It is assumed that ERVs derived from ancient integration events of exogenous retroviruses into the germline. Generally, ERVs are transcriptionally silent and are often defective, differing from the exogenous counterparts by deletions or point mutations that render them unable to produce infectious virus. However, several ERVs maintain at least some intact open reading frames that can be expressed. Particularly, it has been shown that several ERVs containing gag and pol genes also retain the ability to encode functional Env glycoproteins.

Fig. (4).

Maintenance of a reduced cellular and humoral immune response upon JSRV infection and during the course of the virally-induced disease. At the early step of infection, a reduced level of CD4+, the absence of dendritic cells and a low level of circulating specific antibody may be observed. Later, while tumour has developed with alveolar type II cells colonizing the alveolar walls, there is a noticeable influx of macrophages around and in the tumour area, without any other major changes.

The enJSRVs are genetically highly related to JSRV [START_REF] Demartini | Endogenous retroviruses related to jaagsiekte sheep retrovirus[END_REF], which indicates a recent evolutionary relationship [START_REF] Murcia | The transdominant endogenous retrovirus enJS56A1 associates with and blocks intracellular trafficking of Jaagsiekte sheep retrovirus Gag[END_REF]. Several studies suggest that the endogenization of enJSRV occurred 20 My (millions years) in the ancestor species of ungulates [START_REF] Dunlap | Endogenous retroviruses regulate periimplantation placental growth and differentiation[END_REF]. The genomes of normal sheep and goats, as well as several other mammals, contain 15-20 copies of endogenous sequences related to JSRV [START_REF] Hecht | Distribution of endogenous type B and type D sheep retrovirus sequences in ungulates and other mammals[END_REF][START_REF] York | Nucleotide sequence of the jaagsiekte retrovirus, an exogenous and endogenous type D and B retrovirus of sheep and goats[END_REF][START_REF] Carlson | Chromosomal distribution of endogenous Jaagsiekte sheep retrovirus proviral sequences in the sheep genome[END_REF]. Some of these enJSRV are transcriptionnally active in a wide range of tissues [START_REF] Palmarini | Jaagsiekte retrovirus establishes a disseminated infection of the lymphoid tissues of sheep affected by pulmonary adenomatosis[END_REF]. Tolerance may be acquired by expression of these sequences during in utero development and this may account for the low serological response to JSRV. Among the copies present in the sheep genome, one copy named enJS56A1 is a full-length provirus with complete open reading frame for gag, pol and env. The enJS56A1 is mainly expressed in the placenta, but also at a lower level in the lung, the primary site of JSRV-replication. Interestingly, the enJS56A1 Gag can inhibit production of exogenous JSRV particles in a dominant manner [START_REF] Palmarini | Endogenous betaretroviruses of sheep: teaching new lessons in retroviral interference and adaptation[END_REF]. This variant results from several amino acid replacements which are directly involved in the blocking of assembly of infectious JSRV particles. One explanation for the dominant negative effect of enJS56A1 Gag protein on particle release could be that this protein has a defective interaction with some component of the host cell sorting machinery that is responsible for transport and budding of JSRV. As a whole, the presence of this closely related endogenous version of JSRV could explain part of the tolerance and the low immunological response to infectious JSRV.

The immune response to JSRV is still in debate and needs more investigation to understand the mechanisms hold by this virus, leading to the development of a tumour in a poorly responding immunological environment. Whether this is due to the central tolerance related to endogenous forms or due to the peripheral tolerance induced by more direct JSRV immunosuppressive effects, any new therapeutic approach or vaccine development will have to deal with possible autoimmune inflammatory reactions or tumoral development.

Fig. ( 1

 1 Fig. (1). Tumoral lesions observed during ovine pulmonary adenocarcinoma result from the transformation and accumulation of alveolar type II cells in the alveoli and Clara Cells in the bronchioli. While the normal lung presents large open alveoli (A), the architecture of the parenchyma is disturbed in the tumoral lung. Arrows point the accumulation of airway type II cells along the alveoli walls in a typical tumour.

Fig. ( 2

 2 Fig. (2). JSRV viral genome is typically a dimmer of linear, positive-sense, single-stranded RNA with each strand being 7.5 kilobases in length. The gag gene encodes the matrix, the capsid and the nucleocapsid; the pro gene encodes a protease, the pol gene encodes the reverse transcriptase, RNaseH and integrase proteins; and the env gene encodes the glycoproteins needed for receptor recognition and envelope anchoring. An important feature of the retroviral genome is the long terminal repeat (LTR) regions found on both end of the provirus genome. The LTR plays an important role in initiating viral DNA synthesis and its integration as well as regulating transcription of the viral genes. The JSRV genome also contains an additional gene called "x" of unknown role.

Fig. ( 3

 3 Fig. (3).Signalling pathways activated by JSRV in lung epithelial cells. Primary alveolar type II cells derived from ovine tumours display a proliferative advantage compared to normal cells, with a high telomerase activity leading to inhibition of the replicative senescence. Those cells also show a deregulation of the Pi3K/Akt pathway. These mechanisms are implicated in the tumour development and maintenance. Works done on various cell lines have shown that the MAPK pathways can also be implicated when cells are expressing the envelope protein of JSRV[START_REF] Leroux | Jaagsiekte Sheep Retrovirus (JSRV): from virus to lung cancer in sheep[END_REF].

Current Immunology Reviews, 2012, Vol. 8, No.

Archer et al.

Current Immunology Reviews, 2012, Vol. 8, No. 3 Archer et al.

Current Immunology Reviews, 2012, Vol. 8, No. 3 Archer et al.