Pathobiology of bovine leukemia virus

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Pathobiology of bovine leukemia virus

I Schwartz, D Lévy

To cite this version:

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 altered

productive

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 the

infection are under

_study.

BLV / animal models / leukosis

Résumé ―

_{Pathobiologie}

du virus de la leucose bovine

enzootique.

Le virus de la leucose bovine

enzootique (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 BLV

reste

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

_protéine

d’enveloppe, !

des

provirus

BL V

_{génétiquement}

modifiés et à des animaux

_{transgéniques}

résistants

sont en cours d’étude.

BLVlmodèles animauxlleucémie

*

INTRODUCTION

The bovine leukemia virus

_(BLV)

is a cat-tle retrovirus

_responsible

for bovine enzootic

leukemia. Its

_genetic

structure, sequence and

_{pathogenicity}

_present

_{many similarities} to those of the human T-cell leukemia viruses

_(HTLV-1

and

_{2). Seventy}

_percent

of the infected cattle

_present

a chronic increase 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

a

B-cell

_{lymphosarcoma (Burny}

_{et al,}

_1988).

BLV infection involves a

_large

number of

cattle around the world and the

_prevalence

is

_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 have decided to eradicate the infection

_by

apply-ing

strict

_sanitary

measures.

_Nowadays

lym-phosarcoma

cases are

_rarely

found in most western

_European

countries.

Except

for the rare

_{lymphosarcoma}

lesions,

the

_impact

of BLV on bovine health and

_productivity

is not well known and still raises many

questions.

Our purpose here is to review the

_{epidemiological,}

_pathological

and molecular

_knowledge

about

BLV,

and to stress the

_biological

_aspects

which pro-vide us with an

_original

and

_powerful

model

for

_studying

and

_fighting

retroviral diseases.

HISTORICAL BACKGROUND

The first

_reported

case of bovine leukemia

was 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 and it

_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 were visible

_by

electron

_microscopy

after in vitro

culture

_(Miller

et

al,

1969).

In

1976,

Kettmann et al showed that BLV

_particles

are RNA _exogenous viruses and carry a

RNA reverse

_{transcriptase complex;}

this

finding

allowed the classification of BLV

amongst

the

_oncogenic

retroviruses

BLV PATHOGENICITY

BLV induces a

_persistent

and chronic

infec-tion that affects

_essentially

the B

lympho-cyte

population (Kettmann

et

_{al, 1980;}

_Levy

et al,

1987).

The infection can be transmit-ted

_by

the cellular

_components

of infected bovine

blood,

by

infected cultured

cells,

or

_by

free viral

_particles

_produced

in cell culture

(Burny

et

_al,

_1980).

Viraemia is

_only

detectable

_during

the first 2 weeks of infec-tion

_(Portetelle

et al,

_1978).

Thereafter the

expression

of viral

_antigens

is difficult to evi-dence but it can sometimes be detected in the intrafollicular and

_margin

zones of

_lymph

nodes

_{using immunocytochemistry (Heeney}

et

al,

1992).

Cattle

_develop

a

_serological

response to

_capsid

and

_{envelope proteins}

2-8 weeks after inoculation

_(Burny

et

al,

1980).

These antibodies are

_lifelong

and

show

_important

level variations that

proba-bly

relate to the

_{physiological}

and immune status of the animal. Antibodies to viral

reg-ulatory

proteins

(the

Tax and Rex

_proteins)

can also be detected in about 20% of infected cattle

_(Powers

et al,

1991

).

In the years

following

infection,

70% of infected animals show an increase in the

B/T

_lymphocyte

ratio in the

_blood,

with a

seemingly

normal total number of

lympho-cytes (4

000 to 10 000/

_mm

₃

₎

_(Lewin,

_1989).

Among

these

animals,

half

_develop

a

per-sistent

_{lymphocytosis,}

which

_corresponds

to a stable increase in the number of

circu-lating lymphocytes,

sometimes

_reaching

up to 100

000/mm

3

_{(Lewin, 1989).}

The

inte-gration

of the BLV _genome is found in 30%

of the

_{circulating lymphocytes}

and concerns many clones of

lymphocytes (Kettmann

et

al,

1980).

The

_expansion

of the

_lymphocyte

population

results from the

_polyclonal

pro-liferation of mature B

_lymphocytes

which

are

_{cytologically}

and

_{karyotypically}

normal

(Grimaldi

ef al,

1983).

Most of these

lym-phocytes

carry the

CDS,

CDllb and CD11c c

markers

_(Letesson

etal,

1991).

In

mice,

it

seems that the CD5+B-cells constitute a

B-lymphocyte population

of a different

_lineage

from the CD5- B-cells that could be involved in the

_synthesis

of autoantibodies

(Hayakawa

et

al,

1985).

This

_type

of

lym-phocyte

is also found in chronic

_lymphoid

leukemia in humans

_(De

la Hera

et al,

1988).

Are CD5+

_{B-lymphocytes}

the

_preferential

target

for BLV infection?

_Using

fluorescence activated cell

_sorting,

we showed that BLV is 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

_{lymphoproliferative}

potential

of BLV remain to be determined. The ultimate and rare

_expression

of BLV

infection is the emergence of a multicentric

lymphosarcoma

that occurs 1-8 _years after

infection,

in about 1-5% of the animals

(Burny

et al, 1980; Lewin,

1989).

Two-thirds of 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

_mostly

monoclonal for BLV

_integration

and the virus does not seem to

_{display preferential}

inte-gration

sites among tumors from different

animals

_(Kettmann

etal,

1983). According

to some

authors,

the tumor cells are

pre-B-lymphocytes

that are characterized

_by

the lack of

_expression

of the

_{immunoglobulin}

light

chain

_(Van

den Broecke

et al,

1988);

for

others,

they

are mature

_{B-lymphocytes}

showing

an

_isotypic

commutation in the

immunoglobulin

gene

(Heeney

and

Valli,

1990).

Most

often,

the CD5 marker is

expressed

on the tumor cells

_(Depelchin

et

al, 1989;

Letesson

et al,

1991

). This tumoral

form of BLV

_only

affects adult cattle that are over 2 years of age, with an incidence

_peak

around the _age of 5-8 _years

_{(Lewin, 1989).}

This has to be

_{distinguished}

from the bovine leukemia

_sporadic

cases that affect animals

under 1 _year of _age and that are not

BLV TRANSMISSION

Natural transmission

In natural

conditions,

only cattle,

zebus,

buf-falos and

_capybaras

have been found to be infected

_(Burny

ef al,

1980).

The virus is transmitted

_by

infected

lym-phocytes.

About 1 500

_lymphocytes

from a cow with

_{persistent lymphocytosis}

_(0.1

_!I

of

_blood)

are sufficient to infect another ani-mal

_(Mammerickx

et al,

1988).

Most of the

time,

contamination is

_iatrogenic

and occurs

when the animals are

_manipulated

without the

_respect

of

_hygiene

recommendations for

_injections,

_horning,

_tattooing

or rectal examinations

_(Burridge

and

Thurmond,

1981

). In

some areas, transmission

by biting

insects such as Tabanides has been

docu-mented

_(Oshima

et al,

1981

Bronchial

secretions

_containing

infected

_lymphocytes

can be a source of infection

_especially

when

the animals are maintained in crowded

housing (Burridge

and

_{Thurmond, 1981).}

_).

BLV does not seem to be

_sexually

trans-missible

_(Valikhov

et al,

1984).

Calves can

be infected

_during

the second half of

ges-tation,

mainly

when the mother is in

lym-phocytosis (Burridge

and

Thurmond,

1981).

).

Although

potentially

virulent,

the colostrum is 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}

used for

_experimental

infection with BLV.

Indeed,

sheep

are

_highly

sensitive to BLV infection with infected bovine

_lymphocytes

or with viral

_{particles (Mammerickx}

_{et al, 1988).}

Sheep develop lymphosarcomas

in more than 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

_higher

sensitivity

of

_sheep

to BLV

_{pathogenicity}

is related to a

_{higher permissiveness}

of ovine

cells to viral

_{replication (Burny}

_{et al, 1980).}

Ovine

_intraspecies

transmission is

_extremely

rare in natural conditions and are cases of in utero or accidental transmission

_(Bumy

et

al,

1980).

Goats are sensitive to BLV

infection,

but

only

2 out of 24 infected animals

_developed

lymphoid

tumors in 10 years

(Burny

et

_al,

1980).

BLV infection in rabbits is an

_interesting

experimental

model as rabbits

_develop

an

immunodeficiency syndrome

45-763 d after

infection

_(Altanerova

et al,

1989).

After

experimental

infection,

antibodies to cap-sid

_protein

appear in 3 weeks and the

inte-grated

provirus

is detectable in

_peripheral

blood

_{leukocytes (Altanerova}

et al,

1989).

In chicken infected at

birth,

leukemia

development

was observed in 4 chickens out of 88 and BLV _genome could be detected in the tumoral cells

_although

there

was no detectable seroconversion

(Altane-rova

et al,

1990).

Other

_species,

such as rat,

pig,

rhesus

monkey

and

_chimpanzee

have been found to

_develop

antibodies

_following

experimen-tal

infection,

but the

_integration

of BLV

provirus

in host cells was not

_investigated

(Burny

et al,

1980).

Therefore,

one does not

know whether the seroconversion

repre-sents an immune response to a

_replicative

infection or a

_serological

reaction to the intro-duction of viral

_antigens.

According

to

_serological

studies,

humans do not seem to be sensitive to BLV infec-tion

_(Oshima

_{et al, 1981}

_However,

human cells are sensitive to BLV infection in vitro

(Derse

and

Martarano,

1990).

Besides,

hybridization

with BLV-derived

_genomic

sequences was found in 3 cases of human

cases of

_multiple

sclerosis

_(Clausen

et

al,

1990).

However we should be careful in

interpreting

these results because different viruses can show crossed

_genetic

and

sero-logical

reactivities

_(Maruyama

et

al, 1989;

Battles et

al,

1992).

More extensive

epi-demiological

studies should be undertaken in order to

_definitively

rule out the risk of

infection for consumers and

_{professionals.}

SANITARY AND ECONOMIC IMPACT OF BLV INFECTION

BLV is not _very

_pathogenic

or infectious in natural conditions.

_Only

minor economical losses are related to BLV-induced tumors.

However,

the _average

_{dairy production}

of

animals with

_{persistent lymphocytosis}

is 3-10% lower than that of the herd and it

can therefore lead to

_{early culling}

_(Brenner

et al, 1989;

Da

et al,

1993).

Moreover,

cat-tle with

_{lymphocytosis}

seem less able to

_get

rid of some herd infections

_(Brenner

et

al,

1989),

and losses linked to carcass removal when tumors are discovered can be

dra-matic at the herd level. In the United

States,

the economic loss due to BLV infection was

estimated to 86 million dollars in 1992

_(Da

et

al, 1993).

Considering

the transmission mode of the

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,

France had to

_{proceed similarly}

for the sake of EEC

commercial

_{exchanges, although}

seizures for

_{lymphosarcoma}

were

_only

_{2.5 per 100}

000.

_Anyhow,

south-western and north-eastern France were

_particularly

affected

regions,

with 25.4% of

_seropositive

animals

(55%

of the

_herds)

in the Landes area. The tumoral form of bovine leukemia was declared as a

_{&dquo;legally contagious reputed}

disease&dquo; in the 8

_May

1981 decree. This

implies

the

_{systematic culling}

and removal

of carcasses with tumoral lesions and the

marking

of

_seropositive

animals with

_culling

for

_consumption

within 6 months. At that

time,

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}

for

seropositive

animals. In order to

encour-age breeders to

_get

rid of their

_seropositive

animals,

financial

_grants

were

_given

as an

allowance of 2 450 French francs per

ani-mal culled within the month

_following

its identification. Overall these measures cost 394 million francs in 1988. This eradication program was

_expensive

but,

in the

_long

run, it was _necessary in order to facilitate the sale of French cattle on the

_European

mar-ket.

PREVALENCE OF BLV INFECTION IN THE WORLD

Commercial

_exchanges

of

_reproductive

cat-tle led to the

_spread

of the infection around the world.

However,

the

seroepidemiolog-ical status of the infection remains unknown

in a

_large

number of countries.

_Moreover,

regarding

the way the virus is

transmitted,

the infection

_unevenly

affects different

regions

within a

_country,

and different farms

within a

_region.

In France

The mean infection rate was 0.075% in

1992,

whereas it was 2% in 1981

_(Bulletin

tpid6miologique

V6t6rinaire, 2078,

June

1993,

Minist6re de

_{I’Agriculture}

et de la

Pbche, Paris,

France).

However, great

vari-ations remain between

_regions.

In

1992,

the

infection rates of the eastern and south-western French livestocks were over 5%.

Therefore,

even

_though

the BLV

_sanitary

situation

_concerning

BLV has _very much

live-stock is not free of enzootic bovine leukemia

yet.

In

1992,

10 928 infected cattle were culled

_during

the

_{seroepidemiological}

screenings

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 established

fighting

programs

against

BLV. BLV infection

has been eradicated in

_Belgium,

Ireland,

Luxembourg

and

_Norway

and is on its _way to eradication in other western

_European

countries. It does exist as an enzootic form in eastern

_European

countries such as

Alba-nia,

Bulgaria, Yugoslavia

and

_Poland,

espe-cially

in the Baltic countries

_(Latvia,

Esto-nia,

Lithuania) (Sante Animale

Mondiale en

1992,

Office International des

_Epizooties,

Paris,

France).

In North America and Australia

United

_States,

Canada and Australia are

strongly

affected

_by

BLV infection. In USA and

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

the

virus and there are

_important

variations

between states or

_{provinces (Da}

et

al,

1993).

In North

America,

bovine leukemia cases

do not have to be declared.

In

Queensland, Australia,

the

_prevalence

of the infection in the

_dairy

livestock is 13.7%

_(Sante

Animale Mondiale en

1992,

Office International des

_Epizooties,

Paris,

France).

A

_sanitary

program has been

established. In New

_Zealand,

2.4% of the herds are infected

_(Santé

Animale Mondiale

en

1992,

Office International des

_Epizooties,

Paris,

France).

In Africa

Some African countries have estimated the

BLV infection rate and came up with a rather

high

prevalence:

37% in West Africa

(Wal-rand et

al,

1986),

10% in

Guinea,

20% in

Zimbabwe,

and 50% in some

_great

farms in Malawi

_(Sant6

Animale Mondiale en

1992,

Office International des

_Epizooties,

Paris,

France).

BLV infection is thus

_widely

_spread

around the world.

_{Prophylaxis by}

_simple

sanitary

measures based on

_culling

of the

seropositive

animals allowed a dramatic

reduction in the infection rate in western

Europe.

However,

when the infection is endemic 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

to the Oncovirinae

_family.

BLV is _very similar to human 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

_integrated

BLV is 8 714 nucleotides

_{long (Sagata}

et

al, 1985).

Like all

retroviruses,

the

extremi-ties of the

_integrated

BLV

_present

a sequence called LTR

(long

terminal

_repeat)

which is

_composed

of 3 consecutive

_regions

named

U3,

R and U5. The U3

_region

is the most 5’

_region

and includes

_{transcriptional}

regulatory

sequences. The viral mRNAs are

initiated at the first base of the R

_region.

At least 7

_alternately

_spliced

RNAs have been

(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 R

region corresponds

to the

_{polyadenylation}

site

_{(fig 1}

_).

Viral mRNAs

Transcription

of BLV _genome leads to at least 7 viral mRNAs obtained

_by

alternate

splicing (fig 1).

The

_unspliced

mRNA

encodes for the

_{capsid proteins,}

the reverse

transcriptase

and the viral

_protease.

A

monospliced

viral RNA codes for the

enve-lope glycoproteins,

and a

_{doubly spliced}

RNA codes for 2

_{transcription regulatory}

proteins

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 and thus

_regulates

the

_synthesis

of the viral structure

_proteins.

_{Two other mRNAs} pre-sent orf

_encoding

for 2 novel

_proteins

whose role have

_yet

to be

defined,

the RIII and GIV

proteins.

Viral structural

_proteins

BLV viral

_particles

are constituted of 2 viral RNAs and of structural

_proteins

that include the

_{capsid proteins,}

the

_envelope

_proteins,

the reverse

_{transcriptase}

and the viral

pro-tease encoded from the gag, env,

pol

and

prt orf respectively.

Gag, pol

and prt

orf

_products

The

_{capsid proteins,}

reverse

_{transcriptase}

and

_protease

are obtained after

_cleavage

of

3 different

_protein

_precursors initiated from a common AUG located at the 5’ end of the

gag

region.

These 3 _precursors are made

from 3 different

_reading

frames,

and are the

result 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

protein

and is associated both to _the

cap-sid

_proteins

and to the

lipid bilayer

of the

viral membrane

_(Yoshinaka

et

al,

1986).

The mature

_{pol product}

₍₇₀

_kDa)

_is pro-duced from a 145 kDa _{precursor. It}

_presents

a

_{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 an

integrase activity (Burny

etal,

1980).

The

_{prt is synthesized}

from a 65 _kDa

pre-cursor which leads after

_cleavage

to a 14 kDa dimeric

_{aspartic protease.}

It

_performs

the

_cleavage

_{of the gag,}

_prt,

_pol

and env precursors

(Katoh

et al,

1989a).

Env orf

_products

The mature

_gp51

and

_gp30

_{glycoproteins}

are obtained from the

_cleavage

of a 72 kDa

glycosylated

precursor

by

the viral

_protease.

Gp51

is found at the surface of the viral membrane and ensures the

_recognition

of the cellular viral

_receptor

_(Vonbche

et

al,

1992a).

The N-terminal

_segment

of

_gp30

is inserted in an

_oblique

orientation into the

lipid bilayer

and anchors the

_gp51/gp30

complex

in the viral

_envelope

and the infected cell membrane

_(Von6che

et

al,

1992b).

The

_gp30/gp51

association

_helps

membrane fusion

_during

infection of the host cell and

_syncitia

formation

_(Voneche

et

_al,

1992a).

The

_gp30

_{intracytoplasmic}

_portion

pre-sents a motif

_(tyrosine

X X

_leucine),

which is found in the

_signal

transduction molecules of

the T cell

_receptor

_{(Ç chain)}

and of the B cell

_receptor

_(iga

and

_{Ig(3 chains).}

The stim-ulation of a chimeric molecule made of the

CD8

_{extracytoplasmic portion}

and the

_gp30

intracytoplasmic

domain with CD8 anti-bodies induces the

_{phosphorylation}

of cel-lular

_proteins

and a calcium influx into the cell

_(Alber

et al,

1993).

Thus, gp30

may

have a

_signalling

function in the infected cell and anti-BLV

_envelope

_{antibodies may} be able to stimulate

_gp30

and

_dysregulate

the

infected 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 env

nucleotide sequences of 7

_European,

Amer-ican and

_Japanese

BLV isolates showed a

variability

lower than 6%

_(Mamoun

et

al,

1990)

whereas the nucleotide

_variability

reaches 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,

_formerly

called the

_pX

_region,

codes for at least 4

proteins,

the

Tax, Rex,

Rill and _GIV

pro-teins;

it is

_actually

well documented that the

Tax and Rex

_{proteins participate}

in the reg-ulation of BLV

_expression.

Activation of BLV

_expression

by

the Tax

_protein

BLV Tax

_protein

is a 34 kDa nuclear

phos-phoprotein

which transactivates the

initia-tion of RNA

_synthesis

from the U3

_region

of the viral LTR

_(Katoh

et

al,

1989b).

The U3

_region

includes three 21 base

_{pair (bp)}

elements that are

_responsive

to Tax trans-activation

_{(Derse, 1987).}

These 3 elements

are

_{strongly homologous}

and include an

8-bp

core, which is

_homologous

to the

cAMP-responsive

element

_(CRE).

A mutation in

the CRE in the BLV LTR abolishes trans-activation

_by

the Tax

_{protein (Katoh}

et

_al,

1989b).

In

addition,

a cell nuclear factor

called

_{CRE-binding-protein-2 (CREB2)}

has

been shown to bind to the 21

_{bp regions}

and to be

_implicated

in the transactivation of

the BLV LTR

_(Willems

_{et al,}

_1992b).

The U3

_region

of HTLV-1 also includes three 21

_{bp repeats}

centered on

CRE,

which

as for

_promoter

basal

_{activity (Jeang}

et

_al,

1988).

Neither of the HTLV-1 or BLV Tax

proteins

are bound

_directly

to

_DNA;

this

implies

that cellular nuclear factors are

needed to mediate transactivation

_(Jeang

et

_al,

_1988).

In the case of

HTLV-1,

the Tax

protein

makes a nuclear

_complex

with CREB and with

_activating

_{transcription}

factor-2

(ATF-2)

and increases CREB/ATF-2

_affinity

for CRE in the 21

_bp

units but not for the other

CRE

S

,

indicating

that the CRE

_flanking

nucleotides are

_important

in Tax

transacti-vation

_activity

_(Franklin

et al,

_1993).

The CRE

_flanking

nucleotides may also govern

the

_specificity

of the Tax

_proteins

for their

respective

LTR as the Tax

_protein

of BLV is unable to transactivate HTLV-1 LTR and

vice versa

_{(Derse, 1987).}

Regulation

of BLV

_expression

by

the Rex

_protein

Rex is a 18 kDa nuclear

_{phosphoprotein}

which is involved in the

_{post-transcriptional}

control of BLV

_expression.

The Rex

_protein

allows the accumulation of

_unspliced

or

monospliced

viral

RNAs,

probably by

enforc-ing

their

_stability

_{(Derse, 1988).}

A

_250-bp

sequence located between the

polyadeny-lation

_signal

and the

_{polyadenylation}

site in the 3’ R

_region

of the

LTR,

is _necessary for Rex function

_{(Derse, 1988).}

_{This sequence,} called Rex

_responsive

element

_(RRE),

as

well as its

_secondary

structure,

present

great

homologies

with the

_equivalent

HTLV-1 structure.

_{Interestingly}

_{the HTLV-1 Rex} pro-tein can

_replace

the BLV Rex

_protein

and vice versa

_(Felber

et

al,

1989).

In the

absence of Rex

_{protein, only doubly}

_spliced

RNAs are

_synthesized,

which

_implies

that the

_expression

of virion

_proteins

is condi-tioned

_by

Rex

_protein.

Rill and GIV

_proteins

The

_study

of BLV mRNA differential

_splicing

reveals the existence of 2 mRNAs

_coding

for 2 novel

_proteins,

the Rill and GIV

pro-teins

_{(Alexandersen}

et

al,

1993).

The

expression

level of the GIV RNA correlates with the

_development

of

_persistent

lympho-cytosis.

However these 2

_proteins

have not

been detected in vivo and their role is still

under

_{study. Using expression}

vectors for

GIV and RIII in cell transfection

_experiments,

it could be shown that GIV

_weakly

transac-tivates BLV LTR

_activity

and

_potentiates

Rex

_protein

function whereas RIII inhibits Rex

_protein

function

_(S

Alexandersen,

Work-shop

on the

_Pathogenesis

of Animal

Retro-virus,

La

_{Rochelle, France, 1993).}

Lymphocyte

activation and viral

_synthesis

In vivo mRNAs

_coding

for Tax and/or Rex

proteins

are

_easily

detectable

_by

the

_highly

sensitive

_technique

of reverse

_{transcription}

followed

_by

_polymerase

chain reaction

(RT-PCR) (Haas

et al,

_1992).

_Unspliced

or

monospliced

viral mRNAs are absent or

weakly

expressed,

a

_finding

that fits with the

_paucity

of virus

_expressing

cells detected

by immunocytochemistry (Jensen

et

al,

1990).

BLV infection is therefore

_rarely

replicative

in vivo and the mechanisms that

regulate

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

from

sheep

or cattle with

_{lymphocytosis}

unblocks the viral

_synthesis

after 6 h incubation at

37°C

_(Cornil

et al,

1988).

In our

_laboratory,

we have shown that BLV structural

_protein

synthesis

is

_strongly

stimulated

_following

the activation of T

_{lymphocytes by}

lectins such as concanavalin A or

phytohaemag-glutinin

A

_(Djilali

et al, 1987;

Cornil

et al,

1988).

This

_stimulating

effect is mimicked

by

the

supernatant

of activated

_lymphocyte

cultures which indicates that

_cytokines

are

synthe-sis

_(Cornil

et al,

1988).

The

_identity

of the

cytokine(s),

as well as the cell

_type

respon-sible for virion

_synthesis

_(B

cells, CD8

+

T-cells, monocytes

or

_{granulocytes)}

are

unknown

_(Djilali

et al,

1987).

BLV and cell transformation

The mechanism

_by

which BLV leads to

tumoral

_development

has not

_yet

been

elu-cidated . No

_{rearrangement}

of any known

oncogene

(c-myc, c-myb,

c-erbA, c-erbB,

c-src,

c-Ha-ras,

c-fos and

_c-sis)

could be evidenced in BLV-induced tumors

(Kettmann

et al,

1983).

Moreover,

BLV does not show any clear

_{p.referential integration}

site between tumors, a

_finding

which does not

_support

the insertional

_mutagenesis

hypothesis

(Kettmann

et al,

1983).

How-ever, a number of

_experimental

observa-tions indicate that BLV Tax

_protein,

as well

as HTLV-1 Tax

_protein,

can act as onco-genes. Transfection of BLV Tax

_together

with the Ha-ras _{oncogene renders} rat

embryo

fibroblasts

_tumorigenic

in nude mice

(Willems

et al,

1990).

Moreover,

mRNAs

coding

for Tax

_{(and/or Rex)}

are detectable

in animals with

_persistent

_{lymphocytosis}

or

with tumors whereas mRNAs

_coding

for

structural

_proteins

are not

_(Haas

_{et al,}

_1992).

The

_{transactivating}

_protein

Tax is therefore

the most

_probable

viral element involved in

lymphocyte

transformation.

However,

its

expression

is not

_always

encountered in advanced tumoral

_stages

_(Sagata

et

al,

1985);

Tax could therefore

_play

a role in the initiation of

_{tumorigenesis}

but does not seem to be essential in the maintenance of the

tumoral

_stage.

In the case of transformation with

HTLV-1,

a number _{of cellular genes transactivated}

by

Tax have been

identified,

such as the

interleukin-2

_receptor

_(Inoue

_{et al,}

_1986),

the

_{granulocyte/macrophage-colony}

stim-ulating

factor

_{(Nimer et al, 1989),}

vimentin

(Lilienbaum

et al,

1990),

the

_transforming

growth

factor

_{p (Kim}

_{et al,}

_1990).

The

trans-activation of _{cellular genes} is

_proposed

in

order 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 the

Tax

_{transactivating}

domain

_(a

zinc

_finger

region)

is different from the domain involved in cell transformation

_(Willems

et al,

1992a).

Therefore,

the _way in which BLV _Tax pro-tein leads to

_oncogenesis

_may involve a function other than transactivation. For

instance,

viral

_antigens,

such as the SV40

T

_{antigen (Fanning}

and

_Knippers,

_1992),

the adenoviral E1A

_{antigen (Whyte}

et al,

1989)

and human

_{papillomavirus}

E7

_(Dyson

et

al,

1989),

bind to cellular

_proteins

and modulate or

_abrogate

their functions. The cellular

_proteins

that

_physically

associate

with Tax

_during

cell transformation are

actively sought

and _may reveal the exis-tence of new molecules involved in cell transformation.

Although

Tax is most

_probably

involved in

the

_development

of BLV-induced

lympho-proliferative syndrome,

other viral

_proteins

namely

Rex, GIV,

Rill and even env

pro-teins,

are also

_likely

to

_play

a role in BLV

pathogenicity.

The Rex

_protein

of BLV _may disturb cellular mRNAs

_stability,

as the Rex of HTLV-1 increases the IL-2

_receptor

mRNA half-life

_(White

et al,

1991

The

env

protein

may transduce intracellular

_signals

after stimulation

_by

anti-env antibodies

(Alber

et al,

1993).

The

_possible

role of RIII l l and GIV

_proteins

in BLV

_{pathogenicity}

must be

_{investigated, especially}

for GIV whose mRNA level is

_{high during persistent}

lym-phocytosis

(Alexandersen

et al,

1993).

Therefore,

lymphocyte

dysregulation

induced

_by

BLV infection

_potentially

PROPHYLAXIS OF BLV INFECTION

AND PATHOGENICITY

As mentioned

above,

it is

_important

to

develop fighting strategies against

BLV in countries where the

_spread

of the infection is too

_high

to undertake a

_sanitary

prophy-laxis.

Moreover,

BLV

_represents

a

_good

ani-mal model for

_studying

the

_prophylaxis

of

retrovirus diseases.

Indeed,

its

_complex

genetic

structure is

_analogous

to the one of

primate

retroviruses,

and its

_{pathogenicity}

in

sheep

is

_{reproducible, frequent}

and

_easily

detectable within

_delays

suitable to

experi-mentation. Classical vaccination

_strategies

have been tested. Innovative

_strategies

via molecular

_targeting

of the viral

_regulatory

proteins

are under

_study

and are discussed

below.

Vaccination with env recombinant vaccine

The humoral immune response to BLV is

protecting

since serum

_{immunoglobulins}

of

infected

_{sheep prevent}

infection of

_healthy

sheep

in an intradermal

_challenge

with

infected

_{lymphocytes (Kono}

et al,

1986).

A

env 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 intravenous

challenge

of infected

_lymphocytes

6 weeks after vaccinal boost. The

_protecting

vector codes for the

_gp51

and

_{gp30 proteins.}

On

the other

hand,

immunization with vectors

coding

for

_gp51

or

_gp30

alone does not induce

_neutralizing

antibodies and is not

protective.

The immune _memory induced

by

this vaccine remains to be studied.

Vaccination with a

_{non pathogenic}

BLV

This

_strategy

relies on the construction of

infectious,

immunogenic,

but

_{non-pathogenic}

BLVs. BLV mutants with deletions in various genes have been constructed

(Willems

et

al,

1993).

Since BLV

_{proviruses injected}

intradermally

in cationic

_liposomes

are infec-tious in

_sheep,

the infectious and

_pathogenic

potentials

of mutant

_proviruses

could be tested

_(Willems

etal,

1993).

The infectious

potential

is altered for gag,

pol

and env v

mutated

_proviruses

but not for mutants in RIII and GIV. The

_pathogenic

_potential

of RIII and GIV mutants remains to be

estab-lished. In case these mutants are not

pathogenic, they

may be used as infectious

but

_protective

vectors

_against

a wild

_type

virus.

For the same purpose, a chimeric

provirus

made of BLV _gag,

_{pol and}

env genes under the control of

_heterologous

LTRs derived from the murine

_spleen

necro-sis virus was constructed and turned out to be

_replicative

in vitro

_(Temin,

_1993).

The

infectiosity

and

_protection

conferred

_by

this chimeric virus devoid of

_regulatory

_proteins

remain to be tested.

Selection of

_naturally

resistant animals

lmmunogenetic

studies have shown that class-1

_{major histocompatibility complex}

alleles are associated with resistance or

sensitivity

to the

_development

of a

persis-tent

_{lymphocytosis, depending}

on the race

of the cattle. For

instance,

in Holstein

herds,

a relative

_sensitivity

to

_persistent

lympho-cytosis

is associated with the BoLA w8-1 allele

_(Lewin

et

al,

1988).

On the other

hand,

in Shorthorn

herds,

it was found to be associated with the BoLA DA 12-3 allele

(Lewin,

1986).

However the

_development

of

_persistent

_{lymphocytosis}

is

_strongly

asso-ciated with the DRB2 and DRB3 alleles of the class-2

_major

_{histocompatibility}

com-plex (Van Eijk

et al, 1992;

Xu

et al,

1993).

These

_reports

indicate that

_major

studies are needed to find the genes

directly

responsible.

Information on _{these genes}

may have

_agronomic

_{applications shedding}

light

on

_genetic

resistance to retroviral infec-tions and/or cancer

_development.

Construction of resistant

_transgenic

animals

Today transgenesis

in cattle is

_possible.

Constructing transgenic

animals that

express molecules aimed at

_conferring

resis-tance to BLV is conceivable. In _this

per-spective,

a

_ribozyme

that cleaves the Tax/Rex

_coding

RNA has been constructed

(Cantor

et

al,

1993). Ribozymes

are

com-plementary

RNA sequences to a

_target

RNA and are made of a

_{hammer-shaped}

_region

endowed with a

_{catalytic activity.}

An effi-cient

_ribozyme

to the RNA

_coding

for BLV Tax and Rex

_proteins

was shown to reduce the reverse

_{transcriptase}

_{activity by}

92% in

a BLV-infected bat

_lung

cell line

_(Cantor

et

al,

1993).

When

_expressed

_{constitutively}

in

transgenic

animals,

such a

_ribozyme

would

prevent

the accumulation of Tax and Rex

coding

RNAs from the _very

_beginning

of infection and would thus interfere with Tax

transactivation.

_{Consequently,}

the virus infection of the

_neighboring

sensitive cells would be

_considerably

slowed down or even

abrogated

and the

_transgenic

_{animals may} then control the infection and resist to the virus

_{pathogenicity.}

In a similar

_perspective,

transdominant

mutants for HTLV-1 Tax and Rex

_proteins

have also been obtained

_(Rimsky

et

al,

1989;

Gitlin

et al,

1991

These

mutants

are inactive in transactivation and

_they

inhibit

wild

_type

Tax and Rex

_{transactivation,}

prob-ably

by interfering

with their

_transport

to the

nucleus. Isolation of such BLV Tax and Rex mutants could be very

interesting

for

con-structing transgenic

BLV-resistant animals.

Transgenic

cattle would

_{constitutively}

express transdominant mutant Tax and Rex

proteins,

which would

_quench

the wild

_type

Tax and Rex

_{proteins, thereby blocking}

viral

replication.

These cattle would then be resis-tant to the

_propagation

and

_{pathogenicity}

of the virus.

It is clear that such

_transgenic

animals

would

_only

be

_experimental

animals that would

_help

to evaluate the

_efficiency

of these

&dquo;gene

prophylaxis&dquo;

molecules. Beside the

cost of these

_transgenic

_{constructions,}

their introduction to the field is not _easy. It is nec-essary to

_keep

in mind the

_potential

risks inherent to _{exogenous gene}

_expression

in

animals,

because the

_sanitary

and zootech-nical consequences are difficult to

_predict.

Nevertheless,

their introduction in some

American farms where the infection is

widespread

could be

_seriously

considered.

CONCLUSION

BLV infection is

_{widespread throughout}

the

world and it

_{significantly}

affects cattle zootechnical

_performances

without _any dra-matic health

_damage.

However,

we still do not know whether BLV

_potentiates

other herd infection

_{symptomatologies, especially}

in the USA and in Africa where BLV infection

is endemic. In _any case, it would be

inter-esting

to _propose a

_cheap

_prophylaxis

related to the BLV-associated loss of

income. For the time

_being,

vaccination

strategies

are still under trial and are not

yet

applicable

on a

_large

scale.

Furthermore,

BLV infection in

_sheep

turns out to be an excellent

_experimental

model for

_studying

the

_complex

_primate

retro-viruses,

as no

_equivalent

model can be

pro-posed

with the murine or avian retroviruses whose

_genomic

structures are much

sim-pler.

BLV

_{pathogenicity}

is

_reproducible

and

readily

detectable in

_sheep,

an animal

eas-ily

amenable to

_{experimentation.}

It is also

ques-tions remain open.

Why

are

_only

CD5+ B sensitive to the

_{lymphoproliferative}

poten-tial of BLV in vivo? What is the real

_impact

of the Tax

_protein

on

_lymphocyte

dysregu-lation? With which molecules does it

inter-act? The answers to these many

questions

will allow us to unveil the cellular molecules

that are essential for the control of

lympho-cyte

homeostasis.

ACKNOWLEDGMENT

The authors are

especially grateful

to B Schwartz for critical

_reading

of the

_manuscript.

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