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Pathobiology of bovine leukemia virusI Schwartz, D Lévy

To cite this version:I Schwartz, D LÉvy. Pathobiology of bovine leukemia virus. Veterinary Research, BioMed Central,1994, 25 (6), pp.521-536. �hal-00902257�

Review article

Pathobiology of bovine leukemia virus

I Schwartz D Lévy

URA-INRA d’Immuno-Pathologie Cellulaire et Moléculaire, École Nationale Vétérinaire dAlfort,7, avenue du Général-de-Gaulle, 94704 Maisons-Alfort cedex, France

(Received 16 March 1994; accepted 25 July 1994)

Summary ― Bovine leukemia virus (BLV) is a retrovirus similar to the human T-cell leukemia virus(HTLV). Most BLV infected animals (70%) develop a B-cell lymphoproliferative syndrome with alteredproductive traits and 1 to 5% die with B-cell lymphosarcomas. Although BLV infection is worid-wide, west-ern European countries have almost eradicated it by slaughtering the seropositive animals. BLV infec-tion remains endemic in many countries including the United States and prophylactic strategies involv-ing recombinant vaccine vectors, genetically modified BLV and transgenic animals resistant to theinfection are under study.

BLV / animal models / leukosis

Résumé ― Pathobiologie du virus de la leucose bovine enzootique. Le virus de la leucose bovineenzootique (BL V) est un rétrovirus semblable au virus leucémogène humain HTLV La plupart (70%)des bovins infectés par ce virus développent une prolifération lymphocytaire B accompagnée d’une dimi-nution des performances zootechniques, et 1 à 5% des animaux meurent de lymphosarcome B. Bien’que l’infection par le BL V soit mondialement répandue, les nations de l’Europe occidentale l’ont pra-tiquement éradiquée par abattage systématique des animaux séro-positifs. L’infection par le BLVreste cependant endémique dans de nombreux pays, comme les États-Unis. Des stratégies de pro-phylaxie faisant appel à la vaccination avec des vecteurs vaccine recombinants pour la protéined’enveloppe, ! des provirus BL V génétiquement modifiés et à des animaux transgéniques résistantssont en cours d’étude.

BLVlmodèles animauxlleucémie

*

Correspondence and reprints

INTRODUCTION

The bovine leukemia virus (BLV) is a cat-tle retrovirus responsible for bovine enzooticleukemia. Its genetic structure, sequenceand pathogenicity present many similaritiesto those of the human T-cell leukemiaviruses (HTLV-1 and 2). Seventy percentof the infected cattle present a chronicincrease in the number of circulating B lym-phocytes, which can lead to a haematolog-ical status called persistent lymphocytosis,and 1 to 5% of the infected cattle develop aB-cell lymphosarcoma (Burny et al, 1988).BLV infection involves a large number ofcattle around the world and the prevalenceis high on the American continent, in Aus-tralia, Africa, and Asia. As herd contamina-tion follows the introduction of an infected

animal, many European countries havedecided to eradicate the infection by apply-ing strict sanitary measures. Nowadays lym-phosarcoma cases are rarely found in mostwestern European countries.

Except for the rare lymphosarcomalesions, the impact of BLV on bovine healthand productivity is not well known and stillraises many questions. Our purpose here isto review the epidemiological, pathological

and molecular knowledge about BLV, andto stress the biological aspects which pro-vide us with an original and powerful modelfor studying and fighting retroviral diseases.

HISTORICAL BACKGROUND

The first reported case of bovine leukemiawas described in 1871 in the Klaipeda area,Lithuania, and mentioned a cow with super-ficial lymph node hypertrophy and

splenomegaly (Burny et al, 1980). There-after other cases were reported, indicating’that the disease was propagating west andit eventually reached the USA. In 1917, Ken-neth showed that the disease was medi-ated by a contagious agent (Burny et al,1980). In 1969, Miller et al discovered that

lymphocytes of cows with persistent lym-phocytosis produced viral particles that werevisible by electron microscopy after in vitroculture (Miller et al, 1969). In 1976,Kettmann et al showed that BLV particlesare RNA exogenous viruses and carry aRNA reverse transcriptase complex; thisfinding allowed the classification of BLVamongst the oncogenic retroviruses

(Kettmann et al, 1976).

BLV PATHOGENICITY

BLV induces a persistent and chronic infec-tion that affects essentially the B lympho-cyte population (Kettmann et al, 1980; Levyet al, 1987). The infection can be transmit-ted by the cellular components of infectedbovine blood, by infected cultured cells, or byfree viral particles produced in cell culture(Burny et al, 1980). Viraemia is onlydetectable during the first 2 weeks of infec-tion (Portetelle et al, 1978). Thereafter theexpression of viral antigens is difficult to evi-dence but it can sometimes be detected in

the intrafollicular and margin zones of lymphnodes using immunocytochemistry (Heeneyet al, 1992). Cattle develop a serologicalresponse to capsid and envelope proteins2-8 weeks after inoculation (Burny et al,1980). These antibodies are lifelong andshow important level variations that proba-bly relate to the physiological and immunestatus of the animal. Antibodies to viral reg-ulatory proteins (the Tax and Rex proteins)can also be detected in about 20% ofinfected cattle (Powers et al, 1991 ).

In the years following infection, 70% ofinfected animals show an increase in theB/T lymphocyte ratio in the blood, with aseemingly normal total number of lympho-cytes (4 000 to 10 000/ mm3) (Lewin, 1989).Among these animals, half develop a per-sistent lymphocytosis, which correspondsto a stable increase in the number of circu-

lating lymphocytes, sometimes reaching upto 100 000/mm3 (Lewin, 1989). The inte-gration of the BLV genome is found in 30%of the circulating lymphocytes and concernsmany clones of lymphocytes (Kettmann etal, 1980). The expansion of the lymphocytepopulation results from the polyclonal pro-liferation of mature B lymphocytes whichare cytologically and karyotypically normal(Grimaldi ef al, 1983). Most of these lym-phocytes carry the CDS, CDllb and CD11c cmarkers (Letesson etal, 1991). In mice, itseems that the CD5+ B-cells constitute a B-

lymphocyte population of a different lineagefrom the CD5- B-cells that could be involvedin the synthesis of autoantibodies

(Hayakawa et al, 1985). This type of lym-phocyte is also found in chronic lymphoidleukemia in humans (De la Hera et al, 1988).Are CD5+ B-lymphocytes the preferentialtarget for BLV infection? Using fluorescenceactivated cell sorting, we showed that BLVis found in CD8+ T-lymphocytes, monocytes,granulocytes, and mostly in B-lymphocytes,with no preference for the CD5+ B-cell pop-ulation (Schwartz et al, 1994). The param-eters involved in the sensitivity of the CD5+B-lymphocytes to the lymphoproliferativepotential of BLV remain to be determined.

The ultimate and rare expression of BLVinfection is the emergence of a multicentric

lymphosarcoma that occurs 1-8 years afterinfection, in about 1-5% of the animals

(Burny et al, 1980; Lewin, 1989). Two-thirdsof the animals with tumors have had a per-sistent lymphocytosis (Burny et al, 1980).The tumors may affect 1 or several super-ficial and/or deep lymph nodes (Burny et al,1980). In a single animal, tumors are mostlymonoclonal for BLV integration and the virusdoes not seem to display preferential inte-gration sites among tumors from differentanimals (Kettmann etal, 1983). According tosome authors, the tumor cells are pre-B-lymphocytes that are characterized by thelack of expression of the immunoglobulinlight chain (Van den Broecke et al, 1988); forothers, they are mature B-lymphocytesshowing an isotypic commutation in theimmunoglobulin gene (Heeney and Valli,1990). Most often, the CD5 marker isexpressed on the tumor cells (Depelchin etal, 1989; Letesson et al, 1991 ). This tumoralform of BLV only affects adult cattle that areover 2 years of age, with an incidence peakaround the age of 5-8 years (Lewin, 1989).This has to be distinguished from the bovineleukemia sporadic cases that affect animalsunder 1 year of age and that are not asso-ciated with BLV infection.

BLV TRANSMISSION

Natural transmission

In natural conditions, only cattle, zebus, buf-falos and capybaras have been found to beinfected (Burny ef al, 1980).

The virus is transmitted by infected lym-phocytes. About 1 500 lymphocytes froma cow with persistent lymphocytosis (0.1 !Iof blood) are sufficient to infect another ani-mal (Mammerickx et al, 1988). Most of thetime, contamination is iatrogenic and occurswhen the animals are manipulated withoutthe respect of hygiene recommendationsfor injections, horning, tattooing or rectalexaminations (Burridge and Thurmond,1981 ). In some areas, transmission by bitinginsects such as Tabanides has been docu-mented (Oshima et al, 1981 Bronchialsecretions containing infected lymphocytescan be a source of infection especially whenthe animals are maintained in crowded

housing (Burridge and Thurmond, 1981). ).BLV does not seem to be sexually trans-missible (Valikhov et al, 1984). Calves canbe infected during the second half of ges-tation, mainly when the mother is in lym-phocytosis (Burridge and Thurmond, 1981). ).Although potentially virulent, the colostrumis a minor source of infection because the

anti-BLV antibodies it contains protect it

(Van der Maaten et al, 1981 ).

Experimental transmission

Sheep have often been preferentially usedfor experimental infection with BLV. Indeed,sheep are highly sensitive to BLV infectionwith infected bovine lymphocytes or withviral particles (Mammerickx et al, 1988).Sheep develop lymphosarcomas in morethan 90% of the cases in the 6 months to 7

years following infection, depending on the

viral load at the time of infection (Mammer-ickx et al, 1988). It seems that the highersensitivity of sheep to BLV pathogenicity isrelated to a higher permissiveness of ovinecells to viral replication (Burny et al, 1980).Ovine intraspecies transmission is extremelyrare in natural conditions and are cases of inutero or accidental transmission (Bumy et al,1980).

Goats are sensitive to BLV infection, but

only 2 out of 24 infected animals developedlymphoid tumors in 10 years (Burny et al,1980).

BLV infection in rabbits is an interestingexperimental model as rabbits develop animmunodeficiency syndrome 45-763 d afterinfection (Altanerova et al, 1989). Afterexperimental infection, antibodies to cap-sid protein appear in 3 weeks and the inte-grated provirus is detectable in peripheralblood leukocytes (Altanerova et al, 1989).

In chicken infected at birth, leukemia

development was observed in 4 chickensout of 88 and BLV genome could bedetected in the tumoral cells although therewas no detectable seroconversion (Altane-rova et al, 1990).

Other species, such as rat, pig, rhesusmonkey and chimpanzee have been foundto develop antibodies following experimen-tal infection, but the integration of BLVprovirus in host cells was not investigated(Burny et al, 1980). Therefore, one does notknow whether the seroconversion repre-sents an immune response to a replicativeinfection or a serological reaction to the intro-duction of viral antigens.

According to serological studies, humansdo not seem to be sensitive to BLV infec-

tion (Oshima et al, 1981 However, humancells are sensitive to BLV infection in vitro

(Derse and Martarano, 1990). Besides,hybridization with BLV-derived genomicsequences was found in 3 cases of human

myeloma (Slavikova et al, 1987), and anti-bodies to BLV capsid proteins were found in

cases of multiple sclerosis (Clausen et al,1990). However we should be careful ininterpreting these results because differentviruses can show crossed genetic and sero-logical reactivities (Maruyama et al, 1989;Battles et al, 1992). More extensive epi-demiological studies should be undertakenin order to definitively rule out the risk ofinfection for consumers and professionals.

SANITARY AND ECONOMIC IMPACTOF BLV INFECTION

BLV is not very pathogenic or infectious innatural conditions. Only minor economicallosses are related to BLV-induced tumors.

However, the average dairy production ofanimals with persistent lymphocytosis is3-10% lower than that of the herd and it

can therefore lead to early culling (Brenneret al, 1989; Da et al, 1993). Moreover, cat-tle with lymphocytosis seem less able to getrid of some herd infections (Brenner et al,1989), and losses linked to carcass removalwhen tumors are discovered can be dra-matic at the herd level. In the United States,the economic loss due to BLV infection wasestimated to 86 million dollars in 1992 (Da etal, 1993).

Considering the transmission mode ofthe virus, it is theoretically possible to erad-icate the infection using sanitary measures.Denmark (1959) and Germany (1963)began a sanitary fight against BLV infec-tion (Burny et al, 1980). In 1981, Francehad to proceed similarly for the sake of EECcommercial exchanges, although seizuresfor lymphosarcoma were only 2.5 per 100000. Anyhow, south-western and north-eastern France were particularly affectedregions, with 25.4% of seropositive animals(55% of the herds) in the Landes area. Thetumoral form of bovine leukemia wasdeclared as a &dquo;legally contagious reputeddisease&dquo; in the 8 May 1981 decree. Thisimplies the systematic culling and removal

of carcasses with tumoral lesions and the

marking of seropositive animals with cullingfor consumption within 6 months. At thattime, farms were also offered a quality label&dquo;livestock free of enzootic bovine leukemia&dquo;.In 1988, measures against BLV were inten-sified with a systematic screening forseropositive animals. In order to encour-age breeders to get rid of their seropositiveanimals, financial grants were given as anallowance of 2 450 French francs per ani-mal culled within the month following itsidentification. Overall these measures cost394 million francs in 1988. This eradication

program was expensive but, in the long run,it was necessary in order to facilitate thesale of French cattle on the European mar-ket.

PREVALENCE OF BLV INFECTIONIN THE WORLD

Commercial exchanges of reproductive cat-tle led to the spread of the infection aroundthe world. However, the seroepidemiolog-ical status of the infection remains unknownin a large number of countries. Moreover,regarding the way the virus is transmitted,the infection unevenly affects differentregions within a country, and different farmswithin a region.

In France

The mean infection rate was 0.075% in

1992, whereas it was 2% in 1981 (Bulletintpid6miologique V6t6rinaire, 2078, June1993, Minist6re de I’Agriculture et de laPbche, Paris, France). However, great vari-ations remain between regions. In 1992, theinfection rates of the eastern and south-western French livestocks were over 5%.

Therefore, even though the BLV sanitarysituation concerning BLV has very muchimproved since 1981, French cattle live-

stock is not free of enzootic bovine leukemia

yet. In 1992, 10 928 infected cattle wereculled during the seroepidemiologicalscreenings and only 2 animals showed lym-phosarcoma lesions (Bulletin Epidemi-ologique Vétérinaire, 2078, June 1993, Min-istbre de I’Agriculture et de la Peche, Paris,France).

In Europe

Several European countries establishedfighting programs against BLV. BLV infectionhas been eradicated in Belgium, Ireland,Luxembourg and Norway and is on its wayto eradication in other western Europeancountries. It does exist as an enzootic formin eastern European countries such as Alba-nia, Bulgaria, Yugoslavia and Poland, espe-cially in the Baltic countries (Latvia, Esto-nia, Lithuania) (Sante Animale Mondiale en1992, Office International des Epizooties,Paris, France).

In North America and Australia

United States, Canada and Australia arestrongly affected by BLV infection. In USAand Canada, 66% of the dairy herds (10.2%of the animals) and 14% of the meat herds(1.2% of the animals) are infected by thevirus and there are important variationsbetween states or provinces (Da et al, 1993).In North America, bovine leukemia casesdo not have to be declared.

In Queensland, Australia, the prevalenceof the infection in the dairy livestock is13.7% (Sante Animale Mondiale en 1992,Office International des Epizooties, Paris,France). A sanitary program has beenestablished. In New Zealand, 2.4% of theherds are infected (Santé Animale Mondialeen 1992, Office International des Epizooties,Paris, France).

In Africa

Some African countries have estimated theBLV infection rate and came up with a rather

high prevalence: 37% in West Africa (Wal-rand et al, 1986), 10% in Guinea, 20% inZimbabwe, and 50% in some great farmsin Malawi (Sant6 Animale Mondiale en1992, Office International des Epizooties,Paris, France).BLV infection is thus widely spread

around the world. Prophylaxis by simplesanitary measures based on culling of theseropositive animals allowed a dramaticreduction in the infection rate in western

Europe. However, when the infection isendemic and affects a majority of animals,culling is not applicable and other protec-tion measures have to be considered.

BLV VIRUS BIOLOGY

BLV is a type C retrovirus which belongs tothe Oncovirinae family. BLV is very similar tohuman retroviruses HTLV-1 and -2 in terms

of its genetic structure, sequence and reg-ulation of expression.

Viral structure

Viral genome

The complete sequence of the integratedBLV is 8 714 nucleotides long (Sagata etal, 1985). Like all retroviruses, the extremi-ties of the integrated BLV present asequence called LTR (long terminal repeat)which is composed of 3 consecutive regionsnamed U3, R and U5. The U3 region is themost 5’ region and includes transcriptionalregulatory sequences. The viral mRNAs areinitiated at the first base of the R region. Atleast 7 alternately spliced RNAs have beenidentified as well as 8 open reading frames

(orf) designated as gag, ptl, pol, env, tax,rex, RIII and GIV (AIeXandersen et al, 1993).The 3’ U3 region includes the polyadenyla-tion signals and the 3’ extremity of the Rregion corresponds to the polyadenylationsite (fig 1 ).

Viral mRNAs

Transcription of BLV genome leads to atleast 7 viral mRNAs obtained by alternatesplicing (fig 1). The unspliced mRNAencodes for the capsid proteins, the reversetranscriptase and the viral protease. Amonospliced viral RNA codes for the enve-lope glycoproteins, and a doubly splicedRNA codes for 2 transcription regulatoryproteins called Tax and Rex. The Tax pro-tein stimulates the initiation of transcription,whereas the Rex protein favors the stabi-lization and the processing of the monos-pliced and the unspliced viral mRNAs andthus regulates the synthesis of the viralstructure proteins. Two other mRNAs pre-sent orf encoding for 2 novel proteins whoserole have yet to be defined, the RIII and GIVproteins.

Viral structural proteins

BLV viral particles are constituted of 2 viralRNAs and of structural proteins that includethe capsid proteins, the envelope proteins,the reverse transcriptase and the viral pro-tease encoded from the gag, env, pol andprt orf respectively.

Gag, pol and prt orf productsThe capsid proteins, reverse transcriptaseand protease are obtained after cleavage of3 different protein precursors initiated from acommon AUG located at the 5’ end of the

gag region. These 3 precursors are madefrom 3 different reading frames, and are theresult of a 1 or 2 nucleotide ribosomal shift

during translation (Haffield et al, 1989).The proteins encoded by the gag gene

are involved in the formation of the viral cap-sid (Yoshinaka et al, 1986). A 45 kDa pre-cursor is first synthesized and is subse-quently cleaved by the viral protease in themature proteins p15, p24 and p12. The p24is the main capsid protein. The p12 consti-tutes the nucleocapsid and is associatedwith 2 viral RNAs. The p15 is a myristilated

protein and is associated both to the cap-sid proteins and to the lipid bilayer of theviral membrane (Yoshinaka et al, 1986).

The mature pol product (70 kDa) is pro-duced from a 145 kDa precursor. It presentsa RNA-dependent DNA polymerase activity(reverse transcriptase) at its N-terminal por-tion and an RNAase H activity at its C-ter-minal portion. The pol product also has anintegrase activity (Burny etal, 1980).

The prt is synthesized from a 65 kDa pre-cursor which leads after cleavage to a 14kDa dimeric aspartic protease. It performsthe cleavage of the gag, prt, pol and envprecursors (Katoh et al, 1989a).

Env orf productsThe mature gp51 and gp30 glycoproteinsare obtained from the cleavage of a 72 kDaglycosylated precursor by the viral protease.Gp51 is found at the surface of the viralmembrane and ensures the recognition ofthe cellular viral receptor (Vonbche et al,1992a). The N-terminal segment of gp30 isinserted in an oblique orientation into thelipid bilayer and anchors the gp51/gp30complex in the viral envelope and theinfected cell membrane (Von6che et al,1992b). The gp30/gp51 association helpsmembrane fusion during infection of the hostcell and syncitia formation (Voneche et al,1992a).

The gp30 intracytoplasmic portion pre-sents a motif (tyrosine X X leucine), which isfound in the signal transduction molecules ofthe T cell receptor (Ç chain) and of the Bcell receptor (iga and Ig(3 chains). The stim-ulation of a chimeric molecule made of theCD8 extracytoplasmic portion and the gp30intracytoplasmic domain with anti-CD8 anti-bodies induces the phosphorylation of cel-lular proteins and a calcium influx into thecell (Alber et al, 1993). Thus, gp30 mayhave a signalling function in the infected celland anti-BLV envelope antibodies may beable to stimulate gp30 and dysregulate theinfected cell proliferation.

Finally, unlike lentiviruses, envelope pro-teins, the BLV gp30 and gp51 are well con-served among the different variants stud-ied. The comparison between the envnucleotide sequences of 7 European, Amer-ican and Japanese BLV isolates showed avariability lower than 6% (Mamoun et al,1990) whereas the nucleotide variabilityreaches around 66% between the env pro-teins of HIV-1 isolates (Goudsmit et al,1991 ).

Regulation of BLV expression

The 3’ portion of the BLV genome, formerlycalled the pX region, codes for at least 4proteins, the Tax, Rex, Rill and GIV pro-teins; it is actually well documented that theTax and Rex proteins participate in the reg-ulation of BLV expression.

Activation of BLV expressionby the Tax protein

BLV Tax protein is a 34 kDa nuclear phos-phoprotein which transactivates the initia-tion of RNA synthesis from the U3 regionof the viral LTR (Katoh et al, 1989b). TheU3 region includes three 21 base pair (bp)elements that are responsive to Tax trans-activation (Derse, 1987). These 3 elementsare strongly homologous and include an 8-bp core, which is homologous to the cAMP-responsive element (CRE). A mutation inthe CRE in the BLV LTR abolishes trans-activation by the Tax protein (Katoh et al,1989b). In addition, a cell nuclear factorcalled CRE-binding-protein-2 (CREB2) hasbeen shown to bind to the 21 bp regionsand to be implicated in the transactivation ofthe BLV LTR (Willems et al, 1992b).

The U3 region of HTLV-1 also includesthree 21 bp repeats centered on CRE, whichare homologous to that found in the BLVLTR; these are essential for HTLV-1 trans-activation by the HTLV-1 Tax protein as well

as for promoter basal activity (Jeang et al,1988). Neither of the HTLV-1 or BLV Taxproteins are bound directly to DNA; thisimplies that cellular nuclear factors areneeded to mediate transactivation (Jeanget al, 1988). In the case of HTLV-1, the Taxprotein makes a nuclear complex with CREBand with activating transcription factor-2(ATF-2) and increases CREB/ATF-2 affinityfor CRE in the 21 bp units but not for theother CRES, indicating that the CRE flankingnucleotides are important in Tax transacti-vation activity (Franklin et al, 1993). TheCRE flanking nucleotides may also governthe specificity of the Tax proteins for theirrespective LTR as the Tax protein of BLVis unable to transactivate HTLV-1 LTR and

vice versa (Derse, 1987).

Regulation of BLV expressionby the Rex protein

Rex is a 18 kDa nuclear phosphoproteinwhich is involved in the post-transcriptionalcontrol of BLV expression. The Rex proteinallows the accumulation of unspliced ormonospliced viral RNAs, probably by enforc-ing their stability (Derse, 1988). A 250-bpsequence located between the polyadeny-lation signal and the polyadenylation site inthe 3’ R region of the LTR, is necessary forRex function (Derse, 1988). This sequence,called Rex responsive element (RRE), aswell as its secondary structure, present greathomologies with the equivalent HTLV-1structure. Interestingly the HTLV-1 Rex pro-tein can replace the BLV Rex protein andvice versa (Felber et al, 1989). In theabsence of Rex protein, only doubly splicedRNAs are synthesized, which implies thatthe expression of virion proteins is condi-tioned by Rex protein.

Rill and GIV proteins

The study of BLV mRNA differential splicingreveals the existence of 2 mRNAs coding

for 2 novel proteins, the Rill and GIV pro-teins (Alexandersen et al, 1993). Theexpression level of the GIV RNA correlateswith the development of persistent lympho-cytosis. However these 2 proteins have notbeen detected in vivo and their role is stillunder study. Using expression vectors forGIV and RIII in cell transfection experiments,it could be shown that GIV weakly transac-tivates BLV LTR activity and potentiatesRex protein function whereas RIII inhibitsRex protein function (S Alexandersen, Work-shop on the Pathogenesis of Animal Retro-virus, La Rochelle, France, 1993).

Lymphocyte activationand viral synthesis

In vivo mRNAs coding for Tax and/or Rexproteins are easily detectable by the highlysensitive technique of reverse transcriptionfollowed by polymerase chain reaction (RT-PCR) (Haas et al, 1992). Unspliced ormonospliced viral mRNAs are absent orweakly expressed, a finding that fits withthe paucity of virus expressing cells detectedby immunocytochemistry (Jensen et al,1990). BLV infection is therefore rarelyreplicative in vivo and the mechanisms thatregulate this viral latency are still elusive.However a non-immunoglobulin plasma fac-tor absent from serum has been shown to

participate in the inhibition of the viral syn-thesis in vivo (Gupta et al, 1984).

The culture of infected lymphocytes fromsheep or cattle with lymphocytosis unblocksthe viral synthesis after 6 h incubation at37°C (Cornil et al, 1988). In our laboratory,we have shown that BLV structural proteinsynthesis is strongly stimulated followingthe activation of T lymphocytes by lectinssuch as concanavalin A or phytohaemag-glutinin A (Djilali et al, 1987; Cornil et al,1988). This stimulating effect is mimickedby the supernatant of activated lymphocytecultures which indicates that cytokines areimplicated in the stimulation of viral synthe-

sis (Cornil et al, 1988). The identity of thecytokine(s), as well as the cell type respon-sible for virion synthesis (B cells, CD8+ T-

cells, monocytes or granulocytes) areunknown (Djilali et al, 1987).

BLV and cell transformation

The mechanism by which BLV leads totumoral development has not yet been elu-cidated . No rearrangement of any knownoncogene (c-myc, c-myb, c-erbA, c-erbB,c-src, c-Ha-ras, c-fos and c-sis) could beevidenced in BLV-induced tumors

(Kettmann et al, 1983). Moreover, BLV doesnot show any clear p.referential integrationsite between tumors, a finding which doesnot support the insertional mutagenesishypothesis (Kettmann et al, 1983). How-ever, a number of experimental observa-tions indicate that BLV Tax protein, as wellas HTLV-1 Tax protein, can act as onco-genes. Transfection of BLV Tax togetherwith the Ha-ras oncogene renders rat

embryo fibroblasts tumorigenic in nude mice(Willems et al, 1990). Moreover, mRNAscoding for Tax (and/or Rex) are detectablein animals with persistent lymphocytosis orwith tumors whereas mRNAs coding forstructural proteins are not (Haas et al, 1992).The transactivating protein Tax is thereforethe most probable viral element involved inlymphocyte transformation. However, itsexpression is not always encountered inadvanced tumoral stages (Sagata et al,1985); Tax could therefore play a role in theinitiation of tumorigenesis but does not seemto be essential in the maintenance of thetumoral stage.

In the case of transformation with HTLV-

1, a number of cellular genes transactivated

by Tax have been identified, such as theinterleukin-2 receptor (Inoue et al, 1986),the granulocyte/macrophage-colony stim-ulating factor (Nimer et al, 1989), vimentin

(Lilienbaum et al, 1990), the transforminggrowth factor p (Kim et al, 1990). The trans-activation of cellular genes is proposed inorder to explain the lymphocyte perturba-tions induced by HTLV-1. The BLV Tax pro-tein can transactivate the c-fos and somato-statin gene promoters (Katoh et al, 1989b).Recent studies have also shown that theTax transactivating domain (a zinc fingerregion) is different from the domain involvedin cell transformation (Willems et al, 1992a).Therefore, the way in which BLV Tax pro-tein leads to oncogenesis may involve afunction other than transactivation. For

instance, viral antigens, such as the SV40T antigen (Fanning and Knippers, 1992),the adenoviral E1A antigen (Whyte et al,1989) and human papillomavirus E7 (Dysonet al, 1989), bind to cellular proteins andmodulate or abrogate their functions. Thecellular proteins that physically associatewith Tax during cell transformation areactively sought and may reveal the exis-tence of new molecules involved in celltransformation.

Although Tax is most probably involved inthe development of BLV-induced lympho-proliferative syndrome, other viral proteinsnamely Rex, GIV, Rill and even env pro-teins, are also likely to play a role in BLVpathogenicity. The Rex protein of BLV maydisturb cellular mRNAs stability, as the Rexof HTLV-1 increases the IL-2 receptormRNA half-life (White et al, 1991 The envprotein may transduce intracellular signalsafter stimulation by anti-env antibodies(Alber et al, 1993). The possible role of RIII l l

and GIV proteins in BLV pathogenicity mustbe investigated, especially for GIV whosemRNA level is high during persistent lym-phocytosis (Alexandersen et al, 1993).Therefore, lymphocyte dysregulationinduced by BLV infection potentially impli-cates several viral molecules and a lot hasstill to be done in order to delineate the spe-cific role of each of them.

PROPHYLAXIS OF BLV INFECTIONAND PATHOGENICITY

As mentioned above, it is important todevelop fighting strategies against BLV incountries where the spread of the infectionis too high to undertake a sanitary prophy-laxis. Moreover, BLV represents a good ani-mal model for studying the prophylaxis ofretrovirus diseases. Indeed, its complexgenetic structure is analogous to the one ofprimate retroviruses, and its pathogenicity insheep is reproducible, frequent and easilydetectable within delays suitable to experi-mentation. Classical vaccination strategieshave been tested. Innovative strategies viamolecular targeting of the viral regulatoryproteins are under study and are discussedbelow.

Vaccination with env recombinantvaccine

The humoral immune response to BLV is

protecting since serum immunoglobulins ofinfected sheep prevent infection of healthysheep in an intradermal challenge withinfected lymphocytes (Kono et al, 1986). Aenv recombinant vaccine vector was con-structed and tested by a Belgian team(Portetelle et al, 1991, 1993). The protec-tion was tested in sheep with an intravenouschallenge of infected lymphocytes 6 weeksafter vaccinal boost. The protecting vectorcodes for the gp51 and gp30 proteins. Onthe other hand, immunization with vectorscoding for gp51 or gp30 alone does notinduce neutralizing antibodies and is notprotective. The immune memory inducedby this vaccine remains to be studied.

Vaccination with a non pathogenic BLV

This strategy relies on the construction ofinfectious, immunogenic, but non-pathogenic

BLVs. BLV mutants with deletions in various

genes have been constructed (Willems etal, 1993). Since BLV proviruses injectedintradermally in cationic liposomes are infec-tious in sheep, the infectious and pathogenicpotentials of mutant proviruses could betested (Willems etal, 1993). The infectiouspotential is altered for gag, pol and env vmutated proviruses but not for mutants inRIII and GIV. The pathogenic potential ofRIII and GIV mutants remains to be estab-

lished. In case these mutants are not

pathogenic, they may be used as infectiousbut protective vectors against a wild typevirus.

For the same purpose, a chimeric

provirus made of BLV gag, pol and envgenes under the control of heterologousLTRs derived from the murine spleen necro-sis virus was constructed and turned out tobe replicative in vitro (Temin, 1993). Theinfectiosity and protection conferred by thischimeric virus devoid of regulatory proteinsremain to be tested.

Selection of naturally resistant animals

lmmunogenetic studies have shown thatclass-1 major histocompatibility complexalleles are associated with resistance or

sensitivity to the development of a persis-tent lymphocytosis, depending on the raceof the cattle. For instance, in Holstein herds,a relative sensitivity to persistent lympho-cytosis is associated with the BoLA w8-1allele (Lewin et al, 1988). On the otherhand, in Shorthorn herds, it was found tobe associated with the BoLA DA 12-3 allele

(Lewin, 1986). However the developmentof persistent lymphocytosis is strongly asso-ciated with the DRB2 and DRB3 alleles ofthe class-2 major histocompatibility com-plex (Van Eijk et al, 1992; Xu et al, 1993).These reports indicate that major histo-compatibility complex genes are associatedwith sensitivity to BLV, but more extensive

studies are needed to find the genes directlyresponsible. Information on these genesmay have agronomic applications sheddinglight on genetic resistance to retroviral infec-tions and/or cancer development.

Construction of resistant transgenicanimals

Today transgenesis in cattle is possible.Constructing transgenic animals thatexpress molecules aimed at conferring resis-tance to BLV is conceivable. In this per-spective, a ribozyme that cleaves theTax/Rex coding RNA has been constructed(Cantor et al, 1993). Ribozymes are com-plementary RNA sequences to a target RNAand are made of a hammer-shaped regionendowed with a catalytic activity. An effi-cient ribozyme to the RNA coding for BLVTax and Rex proteins was shown to reducethe reverse transcriptase activity by 92% ina BLV-infected bat lung cell line (Cantor etal, 1993). When expressed constitutively intransgenic animals, such a ribozyme wouldprevent the accumulation of Tax and Rexcoding RNAs from the very beginning ofinfection and would thus interfere with Taxtransactivation. Consequently, the virusinfection of the neighboring sensitive cellswould be considerably slowed down or evenabrogated and the transgenic animals maythen control the infection and resist to thevirus pathogenicity.

In a similar perspective, transdominantmutants for HTLV-1 Tax and Rex proteinshave also been obtained (Rimsky et al,1989; Gitlin et al, 1991 These mutantsare inactive in transactivation and they inhibitwild type Tax and Rex transactivation, prob-ably by interfering with their transport to thenucleus. Isolation of such BLV Tax and Rexmutants could be very interesting for con-structing transgenic BLV-resistant animals.Transgenic cattle would constitutivelyexpress transdominant mutant Tax and Rex

proteins, which would quench the wild typeTax and Rex proteins, thereby blocking viralreplication. These cattle would then be resis-tant to the propagation and pathogenicityof the virus.

It is clear that such transgenic animalswould only be experimental animals thatwould help to evaluate the efficiency of these&dquo;gene prophylaxis&dquo; molecules. Beside thecost of these transgenic constructions, theirintroduction to the field is not easy. It is nec-

essary to keep in mind the potential risksinherent to exogenous gene expression inanimals, because the sanitary and zootech-nical consequences are difficult to predict.Nevertheless, their introduction in someAmerican farms where the infection is

widespread could be seriously considered.

CONCLUSION

BLV infection is widespread throughout theworld and it significantly affects cattlezootechnical performances without any dra-matic health damage. However, we still donot know whether BLV potentiates otherherd infection symptomatologies, especiallyin the USA and in Africa where BLV infectionis endemic. In any case, it would be inter-

esting to propose a cheap prophylaxisrelated to the BLV-associated loss ofincome. For the time being, vaccinationstrategies are still under trial and are notyet applicable on a large scale.

Furthermore, BLV infection in sheep turnsout to be an excellent experimental modelfor studying the complex primate retro-viruses, as no equivalent model can be pro-posed with the murine or avian retroviruseswhose genomic structures are much sim-pler. BLV pathogenicity is reproducible andreadily detectable in sheep, an animal eas-ily amenable to experimentation. It is alsoa good model for analyzing the molecularevents involved in cell transformation andtumor progression. However many ques-

tions remain open. Why are only CD5+ B

sensitive to the lymphoproliferative poten-tial of BLV in vivo? What is the real impactof the Tax protein on lymphocyte dysregu-lation? With which molecules does it inter-act? The answers to these many questionswill allow us to unveil the cellular moleculesthat are essential for the control of lympho-cyte homeostasis.

ACKNOWLEDGMENT

The authors are especially grateful to B Schwartzfor critical reading of the manuscript.

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