Infection of primary cultures of mammary epithelial cells by small ruminant lentiviruses

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Infection of primary cultures of mammary epithelial

cells by small ruminant lentiviruses

Catherine Lerondelle, Murielle Godet, J.F. Mormex

To cite this version:

Catherine Lerondelle, Murielle Godet, J.F. Mormex. Infection of primary cultures of mammary

epithe-lial cells by small ruminant lentiviruses. Veterinary Research, BioMed Central, 1999, 30, pp.467-474.

�hal-02695634�

Original

article

Infection of

_primary

cultures of

_mammary

_epithelial

cells

_by

small

ruminant

lentiviruses

Catherine Lerondelle

Murielle

Godet

b

Jean-François

Mornexa

a_{Laboratoire associé de recherches sur les lentivirus chez les}

petits

ruminants, Inra,

École

vétérinaire de _Lyon, 69280

_{Marcy-l’Étoile,}

France

b_{Laboratoire de}

biologie

moléculaire et cellulaire, UMR 49 CNRS,

équipe

Inra 913,

École

normale

_supérieure,

69364

_Lyon

cedex 7, France

(Received 7

_January

1999;

accepted

I8 June 1999)

Abstract-The

_caprine

_{arthritis-encephalitis}

virus (CAEV) and Visna Maedi virus cause

_persistent

infections with

_long

latent

_periods

and induce

_degenerative

and chronic

_inflammatory

lesions of the central nervous _system,

_{joints, lungs}

and udder.

_{Monocyte/macrophage lineage}

is the main _target cell for CAEV and Visna Maedi virus but we

_speculate

that mammary

_epithelial

cells may also be

infected.

_Primary

cultures of milk cells, mammary tissues

of experimentally

and

naturally

infected goats and ewes were used.

_Primary

cultures of mammary tissue from ewes and _goats were infected with the CAEV Cork strain. The lentiviral infection of the

_primary

culture was demonstrated

_by

a typ-ical

_cytopathic

_{effect in mammary}

_epithelial

_{cells and the presence of} an infectious virus in coculture with

_permissive

fibroblasts. To

_identify

the

_epithelial

cells in

_explants

and demonstrate the

anti-genic expression

of CAEV,

primary

cultures were immunostained with

_polyclonal

anti-keratin and monoclonal anti-CAEV

_p30.

Colocalisation studies under a UV fluorescence

_microscope

and

_by

epifluorescence microscopy

showed the

_expression

of

_specific

viral

_antigens

_{in mammary}

_epithelial

cells from the

_eight

_{animals used. Infected mammary}

_epithelial

_{cells may} act as a reservoir for the virus which may

play

an

_important

role in the virus dissemination and in the

_pathogenesis

_{of the mammary} lentiviral disease. © Inra/Elsevier, Paris.

CAEV / mammary

gland

/

_epithelial

cell / lentivirus / small ruminant

*

Correspondence

and

_reprints

Tel.: ₍₃₃₎ 4 78 87 25 93; fax: (33) 4 78 87 25 94; e-mail:

_{c.lerondelle@mail.vet-lyon.fr}

** _{Present address: Laboratoire associ6 Inra/Inserm, communications cellulaires et}

diff6renciation, Inserm U418,

H6pital

Debrousse, 69322

_Lyon

cedex 5, France

Résumé - Infection de cultures

_primaires

de cellules

_{épithéliales}

_{mammaires par les lentivirus} des

_petits

ruminants. Le virus de l’arthrite et de

_{l’encéphalite caprine}

(CAEV) et le Visna Maedi pro-voquent des infections

_persistantes

avec de

_{longues périodes}

de latence et induisent des lésions inflammatoires et/ou

_{dégénératives chroniques}

du

_système

nerveux _central, des articulations,

du poumon et de la mamelle. Les cellules de la

_{lignée monocyte-macrophage}

sont la

_principale

cible des virus CAEV et Visna Maedi ;

cependant

nous faisons

_{l’hypothèse}

_{que les cellules}

_{épithéliales}

mam-maires sont

_également

infectables. Des cultures

_primaires

de cellules du lait et de tissu mammaire de chèvres et de brebis

_{expérimentalement}

et naturellement infectées ont été utilisées. Des cultures

pri-maires de tissu mammaire d’une chèvre et d’une brebis ont été infectées avec la souche de CAEV Cork. L’infection lentivirale des cultures

_primaires

a _{été montrée par l’observation d’un effet}

_cytopathique

caractéristique

dans les cellules

_{épithéliales}

mammaires et la

_présence

de virus infectieux dans les cel-lules en coculture avec des fibroblastes

_permissifs.

Pour identifier les cellules

_{épithéliales}

dans les

explants

et _{pour démontrer}

_{l’expression antigénique}

du CAEV, des

_{immunomarquages}

ont été réa-lisés sur les cultures

_primaires

avec un

_anticorps

anti-CAEV

_p30.

Des études de colocalisation sous

microscope

à fluorescence et _par

_microscopie

à

_{épifluorescence}

ont montré

_{l’expression d’antigènes}

viraux dans les cellules

_{épithéliales}

mammaires des huit animaux étudiés. Les cellules

_{épithéliales}

mam-maires

_{pourraient agir}

comme un réservoir de virus

_{qui pourrait jouer}

un rôle

_important

dans la dis-sémination du virus et dans la

_régulation

de l’infection lentivirale dans la mamelle. © _{Inra/Elsevier,} Paris. s.

CAEV / cellule

_{épithéliale}

/ mamelle / lentivirus /

_petit

ruminant

1. INTRODUCTION

The

_{caprine arthritis-encephalitis}

virus

(CAEV)

and the Visna Maedi virus are

lentiviruses,

non-oncogenic

retroviruses which cause

_persistent

infections with

_long

latent

_periods.

_They

induce

_degenerative

and chronic

_inflammatory

lesions of the

cen-tral nervous _system,

_{lungs, joints}

and udder

!2].

Viral infection of the mammary

gland

is followed

_by

an induration of the udders without individualised nodules but with a

hypertrophy

of the _retromammary

_lymph

nodes. The _mammary tissue shows a

dif-fuse infiltration with

_{lymphoplasmocytes,}

lymph

follicles around the lactiferous ducts and fibrosis

_{[5, 6,}

_18].

The economic costs

of CAEV infection arise from

_{early culling,}

loss of milk

_production,

_poor

_growth

of kids and increased

_replacement

costs

_[13].

The disease also

_increasingly

threatens the _export of live animals.

Cells of the

_{monocyte/macrophage}

lin-eage are the

_major

host cells of the CAEV and Visna Maedi virus. Viral

_replication

in these cells is

_dependent

on cellular differ-enciation so that the

_replication

in

mono-cytes

is restricted

_[2].

A broader _range of cells _may,

_however,

_permit

the

_replication

of CAEV and the Visna Maedi virus. In

vivo,

epithelial

cells of the

_{thyroid, kidney}

and

small intestine of infected _goats were shown

to harbour CAEV RNA. The viral

_antigen

is detected in cells of the central nervous

sys-tem of

_{sheep (including}

_epithelial

cells,

fibroblasts and endothelial

_{cells) [17].}

Ovine aortic smooth muscle cells allow the

repli-cation of Visna Maedi in vitro

_[8].

Another member of the lentivirus

_family,

HIV-1,

can infect

_epithelial

_{cells. The presence of} HIV-I in

_{thymic epithelial}

cells has been demon-strated in vivo and certain HIV-1 strains induce a

_productive

and

_persistent

infection

in these cells in vitro

_[1].

_Epithelial

cells derived from the human cervix

_incorporate

HIV

_proviral

DNA and

_produce

new virus in vitro after infection with HIV-infected T-cells or _monocytes

_{[ 15].}

Infected cells of colostrum and milk are

responsible

for the

_lactogenic

route of CAEV transmission between dams and kids. With HIV and HTLV-1, transmission from the mother to child also occurs

_through

breastfeeding.

These viruses can be derived from blood

_by

infected

_lymphocytes

and

monocytes. It is therefore

_possible

that

non-lymphoid

cells of the breast

_gland

are

pro-ductively

infected.

Indeed,

HIV-1

_replication

has been shown to be _present in

_primary

culture of ductal and alveolar _mammary

epithelial

cells

[16]. Furthermore,

basal mammary

epithelial

cells are

susceptible

to

HTLV infection and transfer infection to

normal

_peripheral

blood

_lymphocytes

_[9].

Polymorphonuclear neutrophils,

lympho-cytes,

macrophages

and

_epithelial

cells are

present in _{goat mammary}

_gland

secretions. In this

_study,

in order to

_investigate

whether

epithelial

cells can be infected with

CAEV,

primary

cultures of _mammary

_gland

tissue were obtained and infected cells were iden-tified

_{by immunostaining}

and colocalisa-tion studies.

2. MATERIALS AND METHODS

2.1. Animals

Milk cells (table I) were obtained from three Saanen _goats

_{experimentally}

infected

_by

intra-mammary inoculation of l06cells infected with the Cork strain of CAEV [7]. After two

lacta-tions, the _goats were killed

_by

intravenous

injec-tion of sodium

_{pentobartital}

_(Dolethal,

Veto-quinol,

Lure, France) and the mammary

glands

were removed

_immediately

after

_slaughter

for mammary tissue culture. The goats were not

lac-tating

and not pregnant at the time of

_slaughter.

Mammary gland

tissues were also obtained from

_naturally

infected animals: two

_freshly

slaughtered

ewes from a local

_{slaughterhouse}

and one _{goat (table I).}

_They

were in mixed

phys-iological

states at

_slaughter.

After

_being

_excised, the mammary

glands

were

_kept

at 20 °C and returned to the

_laboratory

for dissection. All mammary

glands

were free from

_pathological

changes

when examined

_prior

to tissue

_sampling.

2.2.

_Preparation

of

_epithelial

cells 2.2.1. Milk

_epithelial

cells

Cells from defatted milk were cultured in a

modified

_Eagle

medium (MEM) (Seromed,

Poly-labo, France)

supplemented

with 10 % foetal calf

serum _{(IBF, Villeneuve-la-Garenne, France)} as

previously

described _{[ 12].} In order to eliminate all non-adherent cells, the medium was _changed

24 h after inoculation. Cultures were maintained

at 37 °C in a humidified

_atmosphere

of 5 % CO,

and the medium _(MEM-10 °!o _SVF) was

changed

weekly.

2.2.2.

_Primary

_mammary

_epithelial

cells A

_piece

_{of mammary tissue}

_{(approximately}

0.5 x I x I cm) was

_aseptically

excised and

placed

in

_{approximately}

2.5 mL of modified

Eagle

medium, 10 % foetal calf serum

supple-mented with antibiotics:

_penicillin

+

strepto-mycin

( 100 UI/mL + 100

_{yg/mL; Boehringer}

Mannheim, France) and

_fungizone

(1.25

l

ig/mL;

GIBCO _{BRL, France).} The sections were minced

using

scissors, transferred to four-well Lab-Tek slides _{(Nunc, Amilabo, France)} and cultured with 0.2 mL _{of medium per well. The chambers} were filled up with MEM after 24 h of incubation at

37 °C with 5 %

_C0

₂

_.

The medium was

_changed

every 2

days during

the first week of _culture, then

_weekly.

2.3. In vitro infection

of _mammary cell culture

Cells

_growing

from

_explants

_{of mammary}

tis-sue of one ewe and one _{goat (table I)} were infected with the Cork strain of CAEV. After 2 weeks of culture, when the cells covered about 50 % of the lab-Tek slide wells, the cells were

infected at a

_multiplicity

of infection (M.O.I.) of

3 x 10!

_{syncytia forming}

_{units per well. The virus}

inoculum was allowed to adsorb for 8 h and then removed. The cells were washed with medium and incubated for I week in the presence of MEM

_supplemented

with 5 % foetal calf serum.

Uninfected cells were cultured in

_parallel

in the same conditions.

2.4. Detection of viral infection in

primary

culture _{of mammary}

epithelial

cells

After 3 weeks of culture,

typical

viral

cyto-pathic

effects were searched for in milk

_epithelial

cells and cells

_growing

from

_explants

of

mam-mary tissue after a

_{May-Grunwald-Giemsa}

stain-ing.

Milk

_epithelial

cells and cells

_growing

from

explants

were _{examined for the presence of}

infec-tious virus

_by

coculture as

_previously

described

[7]. The cells were cocultured with

_permissive

caprine

fibroblasts and observed

_daily

for the appearance of

syncytia

over a

_period

of 10

_days.

To confirm the

_epithelial

nature of the cells and demonstrate the

_{antigenic expression}

of CAEV, cells

_growing

from

_explants

of

mam-mary tissue were _{analysed by} indirect immunotluorescence after 3 weeks of culture. The cells were washed with _PBS, fixed in cold

(-20 °C) acetone and ethanol _{( I : I , vol/vol),} then rinsed three times with PBS and incubated with

primary

antibodies:

_polyclonal

rabbit anti-ker-atin _(Monosan,

_Genzyme,

_France) and/or

mon-oclonal mouse anti-CAEV

_p30

_(V.M.R.D.,

Pull-man, USA). All antibodies were used

_according

to the manufacturers’ recommendations and diluted in PBS (1:200 for

_{anti-cytokeratin}

and 1:500 for anti-CAEV

_p30).

Lab-Tek slides were incubated for 1 h at 37 °C in a humidified cham-ber. The cells were washed three times in PBS and incubated with

secondary

antibodies: anti-rabbit fluorescein

_{isothiocyanate conjugate}

(Sigma,

France) and/or anti-mouse rhodamine

conjugate

(Jackson, Interchim, France).

Sec-ondary

antibodies ( 1:200) were used

_according

to

the manufacturers’ recommendations. Lab-Tek slides were incubated at 37 °C in a humidified chamber for 1 h.

_Following

extensive

_washings

in PBS, the slides were mounted with a

fluores-cent

_mounting

medium (Dako, France),

exam-ined under a UV fluorescence

_microscope

and then with an

_{epifluorescence microscope.}

Positive and

_negative

controls included the CAEV Cork-infected or -unCork-infected

caprine

fibroblasts derived from

_synovial

membranes.

3. RESULTS

The culture of milk cells included

_single

and small islands

_{of epithelioid}

cells,

and a few

_{macrophage-like}

cells. In

_explant

cul-tures, after 1

week,

several _types of attached cells were

_present:

fibroblasts and islands of small

_epithelial

cells with a small amount

of

_cytoplasm.

Fibroblasts formed net-like bundles

_encircling

the

_epithelial

islands and grew

faster,

covering

more culture surface than did the

_epithelial

cells. Small

epithe-lial cells had the _greatest

_{growth potential}

and _gave

rise,

after 2 or 3 weeks in

culture,

to

_{large squamous-like}

cells with a _very

large

amount

_{of cytoplasm}

and

_frequent

vac-uoles. These

_large

cells

_appeared

in or around the islands of the small

_epithelial

cells. The

_{immunocytochemistry}

with anti-rabbit

_cytokeratin

antibodies confirmed that both small and

_large

_epithelioid

cells from mammary tissue

explants

were

_positive

for

cytokeratin.

In contrast, fibroblasts from mammary

explants

or derived from foetal

goat

synovial

membranes were

_negative

for

cytokeratin.

A _spontaneous

_cytopathic

effect was observed after 3 weeks in milk

_epithelial

cells and in

_primary

culture of _mammary tissue

_{(table I).}

_{In all mammary} tissue

explants,

syncytia

were

_mainly

located in islands of

_epithelial

cells and were smaller in

mammary tissue culture of

naturally

infected ewes

_(three

to five

_nuclei)

than in

experi-mentally

infected

_goats

_(>10

_nuclei)

( figure

I

).

The _{presence of} an infectious virus

induced a characteristic

_cytopathic

effect in

epithelial

cells from _{milk and mammary} tis-sue of

_{experimentally}

infected _goats after

8-10

_days

of coculture with

_permissive

fibroblasts

_{(table I).}

Immunostaining

and colocalisation stud-ies were used to

_identify

infected cells in

pri-mary mammary tissue cultures. The M.O.I. of 3 x

10

3

_{syncytia forming}

_{units per} well induced the

_lysis

of more than 80 % of the cells in

_primary

cultures of the _mammary tis-sue from the ewes.

_Many

of the

_remaining

cells showed an intense

intra-cytoplasmic

staining

for

_cytokeratin

and viral

p30 antigens (figure

2a). Likewise,

a

posi-tive

_{intracytoplasmic}

viral

_{p30 antigen}

stain-ing

is observed in

_epithelial

cells of

experi-mentally

infected _goat

_{explants (figure}

_2b).

In

naturally

infected ewes and the _goat, a few

cells,

identified as

_epithelial

cells stained for

the viral

_{p30 antigen,}

could also be observed with a

_{slighter cytoplasmic staining}

_{(table I).}

In all _mammary tissue

cultures,

mammary fibroblasts never showed CAEV

_p30

anti-gen

staining. Caprine

fibroblasts from syn-ovial membranes infected in vitro with CAEV Cork as

_positive

controls showed red

staining

for the viral

_{p30 antigen}

and no _green

staining

for

_cytokeratin.

The

_specificity

of the

_stainings

was assessed

_by

negative

con-trols where

_staining

was not observed.

4. DISCUSSION

Our

_findings

demonstrate for the first time that mammary

epithelial

cells of ewes

CAEV Cork.

_Moreover,

_they

_{express the} CAEV

_p30

_antigen

in vitro after

experi-mental or _spontaneous infection of _goats and ewes. We

have, however,

not been able

to

_directly

associate

_{permissiveness}

of

mam-mary

epithelial

cells with the release of the infectious virus.

Indeed,

although

the

_explant

cultures contain

_{glandular epithelium,}

fibroblasts,

endothelium and blood

_cells,

milk is devoid of fibroblasts and endothelial cells but not of

_macrophages.

_Furthermore,

the

_macrophages

are the most

_permissive

cell _type for

_supporting

the

_replication

of small ruminant lentiviruses

[3]. Therefore,

one could argue that cultured mammary

epithelial

cells were CAEV

positive only

because

_they

became infected

_by

CAEV

producing macrophages

in milk or

_explant

cultures. There are several _aspects of our

data, however,

that _suggest this was not the case and that the _mammary

_epithelial

cells were CAEV infected in vivo before

_being

cultivated.

First,

CAEV

_replication

in fibro-blasts in vitro is

_rapid

and

_lytic.

However,

we have not observed _any

_cytopathic

effect in fibroblasts of

_explant

cultures. Second,

during

the first week of

culture,

most

macrophages

were eliminated from milk cells and

_explant

cultures

_by

selective

trypsinisation.

Third,

and most

_importantly,

data obtained

_{by immunostaining}

indicated that the

_expression

of the

_capsid

CAEV

_p30

antigen,

a late _stage

_antigen,

occurred

_only

within mammary

epithelial

cells.

Mammary

epithelial

cells _may act as a

reservoir for CAEV in the _mammary

_gland.

It has been established that

_lungs

are a

reser-voir for the Visna Maedi virus. In situ PCR and

_{hybridisation}

have

_previously

demon-strated the presence of Visna Maedi virus in bronchiolar

_epithelial

cells in the

_lungs

of infected

_sheep.

The

_majority

of these cells harbour a

_single

_copy of the viral genome in

a

_{transcriptionally}

silent state

_{[ 14].}

Like-wise,

lentiviruses could enter _mammary

epithelial

cells but infectious virus could be

produced only

after cell activation. Such

growth-arrested

cells may

provide

an in vivo

reservoir for the

_persistence

of

_proviral

CAEV and later reactivation of the cells

_by

host

factors,

for

_example

hormones,

may result in the release of infectious viruses.

CAEV and Visna Maedi virus

_replicate

efficiently

in

_non-dividing

cells. Rather than

depending

on cell

division,

_they

_require

acti-vation and/or differenciation of the host cell for

_productive

_replication.

CAEV infects cells of

_{monocyte/macrophage lineage}

and viral

_replication

in these cells is

_dependent

on cellular differenciation so that

replica-tion in

_monocytes

is restricted.

_Monocytes

contain

_{only proviral}

DNA.

_{Transcription}

of viral RNA and

_production

of viral pro-teins and mature virions does not occur until

the _monocytes localise in tissue and mature to

_macrophages

_{[ 11 Normal}

mammary tis-sue is

_{hormone-dependent}

and

_undergoes

structural and functional

_changes

as a con-sequence of variations in the levels of secreted hormones

_{[ 10].}

Profound ultra-structural differenciation of the

_epithelium

has been described

_immediately

before and after

_parturition

_!4].

Virus _recovery in milk and blood _monocytes is increased at

partu-rition and after an induction of lactation

sug-gesting

that hormone levels _may

_regulate

virus

_production

and/or

_infectivity

_{f 12].}

Hor-mones that stimulate DNA

_synthesis

and

consequently

milk

secretion,

also enhance HIV

_replication

_{in mammary}

_epithelial

cells

[ 161.

Smaller

_epithelial

cells observed in

our

_study

have the

_greatest

_growth

poten-tial and

_give

rise to

_larger

cells which

rep-resent the

_terminally

differenciated

_phase

of the cell maturation _sequence. Differen-ciation-activation of infected _mammary

epithelial

cells _may activate _{viral gene}

expression

at _{the end of pregnancy and the}

infectious virus is then

_produced

in the mammary tissue and colostrum

during

lac-togenesis.

Therefore,

to

_study

viral

produc-tion in the mammary

gland,

it would be

bet-ter to use

_small,

undifferenciated cells from

mammary tissue rather than

large,

differen-ciated cells from milk.

_Moreover,

the

explants

allowed the

_analysis

of CAEV

is conserved and the

_{physical relationship}

between different cell _types is

_preserved.

CAEV has

_{traditionally}

been

_thought

to

be _present

_only

in the _promonocytes and

monocytes in the bone marrow and blood of infected

_goats

as a silent infection. Pro-ductive virus

_replication

is associated with maturation and differenciation of

macrophages

in

_target

tissue. Our results show that CAEV is also present in the

mam-mary

epithelial

cells and that these cells express the

antigen

CAEV

_p30

in vivo.

Thus,

CAEV

infection

in the _mammary

gland

could

_{impact directly}

on the _process of

milk-borne transmission of CAEV

infec-tivity

from dams to kids and contribute to

virus dissemination as it occurs with HIV-1 I and HTLV. While much remains to be understood about the events

_surrounding

the

_pathogenesis

of CAEV in the _mammary

gland,

our

_{findings provide}

a foundation to

more

_fully

define the mechanisms involved in the

_migration

of

_leucocytes

to mammary tissue and the

_relationship

between CAEV infection and the hormonal status of the _goat.

ACKNOWLEDGEMENTS

We are

_grateful

to B.

Pouvelle,

Institut Pasteur

_Lyon,

France for the use of the

epi-fluorescence

_microscope.

We also thank J.M.

_Aynaud,

V. Lambert and C. Le Jan for critical review of the

_manuscript.

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