Antigenic variability of porcine reproductive and respiratory syndrome (PRRS) virus isolates. Influence of virus passage in pig

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Antigenic variability of porcine reproductive and

respiratory syndrome (PRRS) virus isolates. Influence

of virus passage in pig

Alain-Hervé Le Gall, Emmanuel Albina, R. Magar, J.P. Gauthier

To cite this version:

Original

article

Antigenic variability

of

_porcine

_reproductive

and

_{respiratory syndrome}

_(PRRS)

virus isolates.

Influence

_{of virus passage in}

_pig

A

Le

Gall

E

Albina

R

_Magar

JP Gauthier

t _UR

virologie

immunologie

porcine,

CH!’f;, BP

53, 22 440

Plo

q

fha gan,

Frunee;

2

Lahoratoire

_{d’laybiène}

vétérinuire et alimentaire,

Agriculture

Canada,

3400 Cusavant

Ouest,

Saint-Hvacinthe, PQ

J2S

NE3,

Cnnada;

3_{Luboratoire de recherche de la chuire de}

zoologie,

/!!<:<,

domaine de Ici Motte uu Vicomte, BP29, 35!50 Le Rheu, France

(Received

16 October 1996;

accepted

21

January

1997)

Summary &horbar;

In order to

_study

the

_{antigenic variability}

of

_{porcine reproductive}

and

_respiratory

syn-drome virus

(PRRSV),

18

_European

and Canadian field isolates were

_analysed

with a

_panel

of IS

mon-oclonal antibodies

(MAbs)

raised

_against

five different

_European

and one Canadian PRRSV iso-lates. The

_antigenic

_pattern

of these isolates was used to infer their

_{phylogenetic relationships.}

Isolates which had the same

_reactivity

_pattern

were

_{gathered together}

so that five

_{antigenic profiles}

were

_analysed.

The

_pairwise

distances between _{these groups} were defined based on

_antigenic

_pattern

differences. Two main

_antigenic

_groups were obtained,

discriminating

between the

_European

and the Canadian

_populations,

as illustrated

_by

the

_great

distance observed between

_European

and Cana-dian isolates

_(D

= 0.5619 ±

_0.0625)

_compared

to the distance between

_European

isolates

(D = 0.1524 ± 0.0735).

The distance matrix allowed also the construction of a tree

_{diagram. Bootstrap}

analysia

was

_performed

to test the confidence in the

_branching.

The tree

_diagram

confirmed the dis-tinction between the

_European

and the Canadian PRRSV

_{populations. Antigenic variability}

between

an _{isolate and its progeny recovered after} one or two passages in vivo was examined on six isolates.

It was restricted to the GP3

_protein

of the virus.

arteriviridae /

_{porcine reproductive}

and

_respiratory

_syndrome

virus /

_antigenic

_variability

/ monoclonal

_antibody

/

_pig

Résumé &horbar; Variabilité

_antigénique

du virus du

_syndrome

_{dysgénésique}

et

_{respiratoire porcin}

(SDRP).

Influence du passage du virus chez le porc. Afin d’évaluer la variabilité

antigénique

du virus du

_syndrome

_{dyscéiiésique}

et

_{respiratoire porcin}

(SDRP),

I 8 isolats

_européens

et canadiens ont

*

Correspondence

and

_reprints,

été

_analysés

avec I

_{S anticorps}

monoclonaux

_dirigés

contre

_cinq

souches virales

_européennes

et une

souche virale canadienne. La réactivité de ces isolats avec le

_{panel d’anticorps}

monoclonaux a

per-mis d’établir les relations

_{phylogénétiques}

du virus du SDRP. Les isolats

_{qui partageaient}

les mêmes

épitopea

ont été

_regroupés

_{si bien que}

_{cinq profils antigéniyues}

ont été

_analysés.

Une distance a été définie à

_partir

des différences retrouvées entre deux

_{profils épitopiques.}

La

com-paraison

des distances deux à deux entre les isolats

_européens

et canadiens

(D

= _0.5619 ±

_0.0625),

et

les seuls isolats

_européens

(D

= 0.1524

± 0.0735)

a mis en _{évidence deux groupes}

_{antigéniques}

dis-tincts,

l’un

_{d’origine européenne}

et l’autre

_d’origine

américaine. La matrice de distances a _aussi

per-mis d’établir un arbre

_{phylogénétique.}

La solidité des branches a _{été testée par}

_{rééchaiitillonage}

des données

_(boostrap).

L’arbre

_{phylogénétique}

a confirmé l’existence des deux groupes

_{antigéniques}

pré-cédemment établis. La variation du

_{pmfil épitopique}

du virus du SDRP

_après

_{passage chez le porc} a

été étudiée sur six isolats

_{expérimentaux.}

Elle est reliée à la

_protéine

GP3 du virus.

artériviridae /

_syndrome

_{dysgénésique}

et

_{respiratoire porcin}

/ variabilité

_antigénique

/

_anticorps

monoclonal / porc

INTRODUCTION

Porcine

_reproductive

and

_respiratory

syn-drome

(PRRS)

was

first

observed

in

the

United

States of America in

1987

(Zim-merman et

al, 1991),

and then in

_Europe

in 1990. In

_Europe,

the

disease

_{spread rapidly}

from

_Germany

_(Ohlinger

et

al,

1991 )

to the

Netherlands

(Wensvoort

et

al,

1991

),

Bel-gium (Varewijck,

1991),

United

_Kingdom

(White, 1991

Spain

(Plana

Dur6n et

_al,

1992),

France

(Baron

et

_{al, 1992),}

and

Den-mark

_(Botner

et

al, 1994).

It was

_recently

described

and isolated

in

_Japan

_(Shimizu

et

al,

1994; Kuwahara et al,

1994;

Murakami

et

al, 1994).

The

_etiologic

_agent

was

first identified

in the Netherlands

(Wensvoort

et

al,

1 99 1

j.

It is a

_small,

_enveloped,

single-positive-stranded

RNA virus

_(Benfield

et

al, I 992).

The virions are

_pleiomorphic

and have a

spherical shape

(55-65

nm

_diameter)

and a

nucleocapsid

of

30-35 nm.

This

virus con-tains six structural

_proteins:

one

nucleopro-tein

(N),

one

_{non-glycosylated}

membrane

protein

(M)

and four

_glycosylated

mem-brane

_proteins

_{(GP2, GP3,}

GP4 and

_GPS)

(Van

Nieuwstadt et

al,

1996).

PRRS virus

(PRRSV)

is

_actually

a member of the

_newly

proposed

Arteriviridae

_family,

which also includes the

_equine

arteritis virus

_(EAV),

the lactate

_{dehydrogenase-elevating}

virus

and the

simian

_haemorrhagic

fever virus

(SHFV)

(Conzelmann

et

al, 1993;

Meulen-berg et al,

1994).

The

_{antigenic variability}

of the PRRS virus was first

_{recognized by polyclonal}

antibodies

in an

_{immunoperoxydase}

mono-layer

assay (IPMA) and western blot

(Wensvoort et al,

1992;

_{Kwang et al,}

1994).

Polyclonal

antisera were able to

discrimi-nate

between

the

American and

_European

isolates

_(Wensvoort

et

_{ai, 1992).}

This

vari-ability

was then

_using

MAbs

_(Nelson

et

_al,

1993;

_Kwang

et

_{a], 1994,}

Wieczoreck-Khromer et

_{al, 1996;}

Drew et

al,

1995,

De

Kluyver et

al,

1995;

_{Magar et}

al, 1995;

Yoon

et al,

1995a).

The

_objective

of this

_study

was to

pro-vide

a more

_{comprehensive analysis}

of

the

antigenic variability

within the

_European

and

Canadian

viruses

_using

a wide

panel

of

MAbs

from

_Europe

and Canada. One

hybridoma specific

for

PRRSV was pro-duced

and

the others were

_gifts

from other

laboratories.

This paper

also

_reported

the

_antigenic

variation

of PRRSV isolates

observed after

one or two _{passages in}

_pig.

Five PRRSV

isolates

were recovered

from different

_pigs

parental

strain and

_represented

a

_single

pas-sage

in

vivo. Another

isolate

was recovered

after transmission of the virus from a sow to

its

_piglets

and

then

transmission

from

the

piglets

to a

_{contact-exposed pig:}

the virus

isolated from the

_{contact-exposed pig}

rep-resented

the

second

_{passage in}

vivo

of the

virus

inoculated to the sow.

MATERIALS

AND

METHODS

Virus strains

Eighteen

PRRSV isolates were obtained from different

_European

countries and Canada

(tableI).

Isolate III was derived from isolate 11

_through

the infection of sows, transmission to their

off-spring, persistent

infection for l7 to 18 weeks

in the

_piglets,

before

_being

transmitted to

con-tact-exposed pigs

(Albina

et

at, 1994).

_Briefly,

specific pathogen

free

_(SPF)

sows were infected with PRRSV isolate II at 90

_days

of

_gestation

and allowed to farrow.

_{Typical signs}

of the

dis-ease were

_observed,

_including

abortion and still-birth. All

_{surviving piglets developed}

clinical

signs by

4 to _{5 weeks of age and had}

serocon-verted

_by

8 weeks _{of age. When}

_they

were 13 3 weeks of age,

they

were unable to transmit the virus to

_{indirectly exposed}

SPF

_pigs.

Nine weeks later

_(they

had been

_seropositive

for 15

weeks),

they

were

_placed

in direct contact with five SPF

piglets.

Infected

_pigs

were submitted to a corti-costeroid

_{immunosuppression}

for I week and

sera from

_{contact-exposed piglets}

were tested for the presence of PRRSV and antibodies. Three

out of five

_{contact-exposed piglets}

seroconverted within 5 weeks and all

_piglets

were virus

_positive

7 weeks after contact. Isolate III was recovered from the serum of one contact

_piglet.

Isolates 56237, 56239, 56240, 56241, 56243 derived from isolate I. Five

_pigs

were inoculated with 3 mL in each nostril of isolate I

₍₁₀

₂

TCID

5

()/mL).

One week

later,

they

received

intravenously

a booster dose

_{( I ()4}

_TCID

₅

_{( /iiiL)}

of the same isolate. Virus was recovered from sera

of the five

_pigs

5 weeks after infection.

Virus

_purification

Two PRRSV isolates were

_purified

for

produc-tion of

_{specific antibody secreting hybridoma.}

Porcine alveolar

_macrophages

(PAM)

were

prepared

as described

_by

Wensvoort et al

( 1991

).

Briefly,

cells were seeded on 75

cm’-

culture flasks

_(Falcon).

After 4 h of incubation at 37 °C and 5%

_CO2,

_they

were infected with isolate I

or II. When a 70%

_cytopathic

effect was observed, the

_macrophages

were frozen and

thawed twice. The clarified

supernatant

was

centrifugation

at 20

₀₀₀

_{g for}

30 min at

4 °C,

the

_pellet

was

_re-suspended

in TNC

buffer,

then

re-precipitated

for 2 h at 4 °C and

_centrifuged

again.

The

_pellet

was

_resuspended

in TNC

buffer,

laid on sucrose cushions

_(10%,

40%

w/v)

and

centrifuged

at 40

000

g for

3 h at 4 °C. The vis-ible band was collected and

_{dialysed overnight}

against

TNC at 4 °C.

Monoclonal antibodies

Production

_{of MAb}

44H8

The MAb 44H8 was

_produced

in our

_laboratory

against

a

_pool

of two isolates of PRRS virus: I

and II.

_Briefly,

Balb/c mice were inoculated

intraperitonealy

for 5 consecutive weeks with the two

_purified

PRRSV isolates

_(Drew

et

al,

1995).

For the first

_injection,

the

_purified

virus

was mixed with an

_equal

volume of the Freund

complete adjuvant.

For the

_subsequent

three

boosters,

purified

virus was mixed with the

Fre-und

_{incomplete adjuvant}

_(v/v).

The last inocu-lation was

_performed

three

_days

before

spleno-cyte

collection and consisted of an

_{intraperitoneal}

injection

of

_purified

virus in

_phosphate

buffered saline

_(PBS).

The

_spleen

cells were

_hybridized

with

_SP

₂

₀

_myeloma

_{cells in the presence of 50%}

polyethylene glycol

solution

_{(PEG 1500,}

Boehringer

Mannheim,

Germany)

as described

by

the manufacturer.

Culture

_supernatants

of viable

_hybridomas

were screened for the presence of antibodies

_by

IPMA

_(Wensvoort

et

al, 1991 )

and ELISA

(Albina

et

al, 1992)

modified for the detection of mouse antibodies. At the same

time, all the

antibodies from the culture

_supernatants

were

titrated

_by

ELISA to re-test

_hybridomas

that had grown too

_poorly

to

_{produce large}

amounts of antibodies. Selected

_hybridomas

were cloned

three times

_{by limiting}

dilution.

The MAb 44H8 reacted in IPMA, but not in

ELISA

_(data

not

shown).

The IPMA reaction on

isolate I was dark coloured whereas

_only

a small

proponion

of cells infected with isolate II were

weak coloured.

The other MAbs

MAbs other than 44H8 were

_{kindly supplied}

_by

different

_European

and Canadian laboratories.

The

_origin

and

_specificity

of these MAbs are

summarised in table II.

Antibody

detection

Immunoperoxydase monolayer

assay

The

_{immunoperoxydase monolayer}

_assay was

performed

as described

_by

Wensvoort

(Wensvoort

et

_{al, 1991). Freeze/thawed}

super-natants of second or third passage of the different

virus isolates were seeded on PAM in 96 well microtiter

_plates

_(Falcon).

When a 20 to 30%

cytopathic

effect was

_observed,

the cells were

fixed with ethanol 95!/o at 4 °C for 20 min.

Appropriate

dilutions of MAbs were added

to the wells

_containing

the fixed infected PAM. The presence of a

_specific

MAb was revealed

by

rabbit anti-mouse

_{immunoglobulins}

conju-gated

with horseradish

_peroxydase

(Dako)

and its substrate

_{3-amino-9-ethylcarba!ol (Sigma).}

Enzyme

linked immunosorbent

_assay

The two ELISA described were

_only

used for

screening hybridoma

supernatants.

The ELISA for the detection of antibodies

specific

to PRRSV

_{developed by}

Albina was

adapted

for the detection of mouse antibodies

(Albina et al,

1992).

Briefly, virus-specific

anti-bodies in the _supernatants of

_hybridoma

cells

were incubated with

_positive

and mock

_antigens

previously

coated on 96-well microtiter

_plates.

Antibodies were revealed with rabbit anti-mouse

immunoglobulins

conjugated

with horseradish

peroxydase.

The

_peroxydase

_substrate,

orthophenylene

diamine,

was added to each well and reaction was

_stopped

30 min later

_by

addition

of sulphuric

acid.

_Optical

densities were recorded

with a

_{spectrophotometer.}

For the titration of the total amount of anti-bodies in the culture _{supernatants,} rabbit

anti-mouse antibodies were diluted in carbonate buffer

(pH

9.6) and coated

on P96 well microtiter

_plates

for I6 h at 4 °C.

_Supernatants

of

_hybridoma

cells

were

_incubated,

as

_already

described for the first

ELISA. A standard

_consisting

of a _{range of}

puri-fied MAb of

_{pre-determined}

concentrations was

Mapping

method

The PHYLIP

_package

was used for the

_analysis

and the constitution of a tree

(Felsenstein

et

al,

1989).

This

_package

consists of a series

_of

phy-logenic

and

_grouping

_programs.

First

_approach,

the distances between isolates

were established and included in a matrix. The

distance between two isolates was defined as

fol-lows: D = _I -

[n(

I I

+

+ n(00)j

/ K, where

D: distance calculated between two

isolates;

n(

I I):

number of monoclonal antibodies that

react with both

isolates;

n(00):

number of

mon-oclonal antibodies that do not react with both

isolates;

K: total number of monoclonal

anti-bodies

The

unweighted

pair-group

method of arith-metic

_averaging

(UPGMA)

was used to construct a tree

_diagram

(Sneath

et

al,

1973).

In a second

_study,

_{bootstrapping}

was tested to

estimate the confidence that existed between each branch of the first tree

(Felsenstein

et al,

matrixes were then calculated for each set of data

and one tree was constructed for each matrix

_by

UPGMA.

_Finally,

the

_majority

rule consensus

tree was

_generated

from the 500

_preceding

trees.

RESULTS

MAbs

virus

_{binding specificity}

The IPMA

reactivities of

the MAbs are

reported

in table III.

MAbs directed

_against

the

_{nucleoprotein}

n N

of

PRRSV

discriminated well between

the

_European

and the Canadian

PRRSV,

except

for P3/27 and

WBE I . MAbs

directed

against

other

_proteins

were variable on both

continents.

MAbs directed

_against

the

_{nucleoprotein}

N or the membrane associated

non-glyco-sylated protein

M

showed

_stronger

_staining

of

the infected cells than did the other MAbs.

Some

isolates had

the same

_pattern

of

reactivity

in IPMA with all the MAbs tested.

Five groups

of isolates

_having

identical

pat-terns

of

_reactivity

were

determined:

_{1 ) IX,}

I,

56237,

56240,

56241, 56243,

NL4.1,

NY3 and

AV30;

2)

II,

V,

VI,

and

2.72;

3)

NL2.2 and

_{2.25; 4)}

LHVA-92-2

and

_LHVA-94-7;

5)

56239 and

H2. The

other PRRSV

iso-lates

_{(III, IV,}

L51/2/92 and

₉₄₈₀₇₎

had

a

unique

pattern

of

_reactivity

with

all

the

MAbs.

Distances between isolates

Canadian

and

_European

isolates were

_clearly

discriminated

_by

their

_reactivity

in IPMA as

illustrated in table

IV:

the distance

between Canadian and

_European

isolates

was much

_greater

_(D

=

0.5619

_±

_0.0625)

compared

to the distance between

_European

isolates

(D

=

0.1524

±

_0.0735).

This was

particularly

obvious

for the

_{nucleoprotein,}

as

shown in table

III.

Figure

I shows the tree

_diagram

obtained

by clustering

the

overall

distances between

PRRSV isolates based

on their

_reactivity

with this

_panel

of MAbs.

Clusters of

_European

viruses

determined

by

UPGMA

_regrouped

_any

isolates from

any

country

for

any date of

isolation. There

did

not seem to be

_geographic

or

chrono-logical relationship

between the

_European

isolates.

Interestingly,

isolates

which were recov-ered

after

_persistence

in

_pigs

differed from

the

_challenge

isolate

_by

a

GP3

_epitope

rec-ognized by

WBE2. Isolate

III,

which

derives

from isolate

II,

has lost this

_epitope

com-pared

to

its

_parental

isolate

_{(table III).}

In contrast, isolate 56239

contained

this

epi-tope

although

isolate

I,

from which it

orig-inates,

did not.

Bootstrap analysis

reinforced the

_strong

discrimination

between

Canadian

and

Euro-pean isolates: this branch

was

found

_again

in

more than 93% of the 500 trees. Such a

result was

also observed

_{using parsimony}

method

_(data

not

_shown).

_Groups

within

European

isolates

were

much

less

evident

since the most

_{representative}

branch was

only

found

in 64% of

the

trees with UPGMA

and

in less than

50%

with

parsi-mony

method. This branch

separated

group 2 and III from the other

isolates,

but this was not

_{statistically}

_significant.

DISCUSSION

In this

_{study, antigenic}

variations between

Canadian and

_European

isolates of PRRS virus were confirmed. These results are in

agreement

with

_previous

studies

_(Wensvoort

et

_{al, 1992;}

Nelson et

_{al, 1993;}

_Kwang

et

al,

1994,

Wieczoreck-Khromer et

_{al, 1996;}

Drew et

al, 1995;

De

_Kluyver

et

al, 1995;

Magar

et

al,

1995;

Van Nieuwstadt et

al,

1996;

Yoon et

al,

1995a).

They

reinforced

the

_hypothesis

that North-American and

sub-populations

that

_might

have

evolved

separately

from a common or two

indepen-dent ancestors. MAbs

_against

the

nucleo-protein clearly

discriminated

the two

popu-lations,

as

_already

shown

_by

others

_(Nelson

et

at, 1993;

Wieczoreck-Khromer

et

at,

1996;

Drew et

a], 1995;

Dea et

at,

1996).

Some

MAbs

_specific

to

the

_{nucleoprotein}

(P3/27, WBE1)

recognized

isolates of both

European

and Canadian

_origin.

Since P3/27

bound

to

all

other

isolates tested

_(eight

Euro-pean

and 12

North-American;

Wieczoreck-Khromer

et

_{al, 1996) and WBE1}

did

not

_(it

reacts with 24 other

_European,

but

not

with

eight

North-American

_isolates;

Drew et

_at,

1995),

P3/27 could be a

_good

candidate for

diagnostic

tests such as

_competitive

bind-ing

assays

or

_{immunocapture}

of

PRRSV.

On

the

other

hand,

the other MAbs

_specific

to the

_{nucleoprotein}

_(1CH5,

WBE4, WBE5,

WBE6,

126.9)

could be used

to test the

American

or

_European

_origin

of PRRSV.

MAbs

_specific

for N or M

showed

stronger

staining

than

the others because

of

the

_predominance

of these

_proteins

in the virion. This

has been

_already

observed

in immunoblots

(Van

Nieuwstadt

et

_{al, 1996).}

Moreover,

the

_{nucleoprotein}

_{has been}

pre-viously

described

as

_being

the most

immunogenic

protein

of

PRRSV

(Yoon

et

at, 1995b).

This

could

_{explain why}

MAbs

have been

_frequently

_{produced against}

the

nucleoprotein

whereas MAbs

_against

the other viral

_proteins

are more

difficult

to

obtain

(Nelson

et

at, 1993;

Wieczoreck-Khromer et

_at,

1996;

Drew

etai, 1995; Dea

et al, 1996;

Van

Nieuwstadt

et

at, 1996).

MAbs

_specific

for viral

_proteins

other than N or M _gave

non-homogeneous

pat-terns

of

_reactivity

between

American and

European

isolates.

Analysis of antigenic diversity

within the

European

isolates

_using

UPGMA

did

not

permit

the

identification

of

_sub-groups

according

to

_origin

or

date

of

isolation.

When the

_parsimony

method or factorial

analysis

of

_{correspondence}

was

_used,

we

observed

the same

results

(data

not

_shown).

No

_{relationship concerning}

the

_origin

or

period

of isolation could be established

between the

_European

isolates tested.

Interestingly,

GP3 seemed to be

associ-ated with the

_persistence

of PRRSV in

_pigs.

In the two

_examples

observed,

the

differ-ences between the

_original

and derived

iso-lates were

localized

on

the

_epitope

recog-nized

_by

WBE2. ORF3

is

also the most

variable

ORF in

arteriviruses

and the

cor-responding protein

is the most

_potentially

N-glycosylated

viral

_{protein (Murtaugh}

et

al, 1995).

In

PRRSV,

some

_regions

of

GP3

are

_highly

variable

as demonstrated in this

study

and others

(Wieczoreck-Khromer

et

_al,

1996;

Drew et

al, 1995),

but other

_regions

are not

_{(Wieczoreck-Khromer}

et

al, 1996;

Katz et

_{al, 1995).}

_Actually,

some

mono-clonal antibodies

_against

GP3

_recognize

the

epitopes

of

all the

_European

or

American

isolates tested

whereas

others are

_specific

for

_only

some

of

them

(Wieczoreck-Khromer et

al, 1996;

Drew et

al,

1995).

European

field

_origin

antisera

react to the

carboxyterminal

portion

of

GP3,

which is

expressed

in SF-9

_cells,

whereas

American

sera

do

not

(Katz

et

al,

1995).

Antigenic

variation

in RNA viruses is not

_only

a con-sequence

of

immune

selection,

but is also

a

result of their

quasispecies

behaviour and

evolution

_(Domingo

et

_{al, 1993).}

For

this

reason, it

would

be

unlikely

that

changes

in

the

_{epitope specific}

for WBE2

_depended

on

quasispecies

behaviour since the

_antigenic

variation

was

demonstrated

on the same

epi-tope

after

the

_{passage of} two

different

PRRSV isolates in

_pigs.

This variation would

_probably

be

determined

_by

the immune response of the host

against

GP3.

High

glycosylation

and

_variability

of

GP3

might

indicate

that this

_protein

is

_implicated

in virus-host cell

interactions.

One

_important

way of immune

system

intrusion

into

virus-host cell interactions

is

neutralization with

antibodies. Until

now,

there

was no

_proof

that GP3 was involved in neutralization of

et

al,

1996;

Van

Nieuwstadt

et

_{al, 1996).}

However,

WBE2

had

never been

tested in

virus neutralization

tests before. Further studies on GP3

_{epitopes, including}

the

recognition by

WBE2

_might

be

_interesting

for

_{investigations}

on

virus-host

cell

inter-actions and

on

the

means of PRRSV escapes from the immune responses of the host. This

study

could be of

_importance

to understand how

PRRSV,

as

well

as

other

arteriviruses,

can

_persist

in their

host

for

a

_long

_time,

even

in the presence of an immune response

developed by

the host to the virus.

ACKNOWLEDGMENTS

We are

_grateful

to the scientists who

_provided

the viruses and MAbs: T Drew

_(Central

Veteri-nary

laboratory,

Addlestone,

United

_Kingdom),

R

_Magar

(Laboratoire

d’hygiene

vétérinaire et

alimentaire,

Saint-Hyacinthe,

Quebec),

J Plana Duran

(Laboratorios

Sobiino

SA,

Vall de

_Bianya,

Spain),

R Rosell

(Laboratorio

de Sanidad

ani-mal, Barcelona,

Spain),

A Van Nieuwstadt

(Insti-tute for Animal Science and

Health,

Lelystad,

the

_{Netherlands),}

VF

_Ohlinger

and E Weiland

(Federal

Research Centre for Virus Disease of

Animals,

Tubingen, Germany).

We also thank R

_{L’Hospitalier}

for her

_help

in the factorial

anal-ysis

of

_{correspondence.}

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