Archives of Virology 87, 151--161 (1986) Archives of Virology © by Springer-Verlag 1986

Antiflenie and Bioloflieal Charaeterization of Avian Paramyxovirus Type 1 Isolates from Pifleons

By

G. MEULEMANS 1, M. GONZE 1, M. C. CARLIER 1, P. PETIT 1, A. BURNY s, and LE LONG 2

1 N a t i o n a l I n s t i t u t e for V e t e r i n a r y R e s e a r c h ,

Brussels, Belgium, 2 Department. of Molecular Biology, University of Brussels,

I{hode-St-Gengse, Belgium

With 3 Figures

Accepted May 31, 1985

Summary

21 A/PMV-1 viruses were isolated from pigeons and characterized using polyclonal and monoclonal antisera in hemagglutination inhibition, sero- neutralization and immunoprecipitation studies.

Polyclonal and monoclonal antibodies directed against the HN and F proteins of Italien virus reacted with all pigeon isolates showing a close relationship between chicken velogenic and pigeon viruses. Differences in the M.W. of F0, P and M proteins were however observed between pigeon and chicken Italien virus.

Marked differences in virulence were recorded among pigeon isolates; these were reflected by great variation in the IVPI of the different strains.

Introduetion

Since 1982, an outbreak of infection with paramyxoviruses of serotype t (A/PMV-1) occurred in racing pigeons throughout Europe (AL~xA~DEa et al., 1984a).

The disease was reported to be present in I taly (PERI~I et al., 1982; BIA~ClFIORI and FlOlClNI, t983), in the Federal l~epublic of Germany (RICHTER, 1983) in France (GvlTTET et al., 1984), in Great Britain (ALExAN- ])ER et al., 1984a; i984b), in the Sudan (EIzA and OSIER, i984), in Denmark, Portugal, Switzerland, Sweden, Japan, Hungary and Holland (ALEXANDER et al., 1984c).

11 Arch, }'iroI. 87]3--4

152 G. MEULEMANS et al. :

In Belgium, the first case was observed in April 1983 and reported by VIAblE et al. (1983). Until July 1983, no other case was seen. At that time, the disease became epizootic and 21 A/PMV-1 viruses were isolated in our laboratory from affected pigeons during a 4 month period.

In this paper, we report on the antigenic characterization of these pigeon A/PMV-1 viruses using monoclonal antibodies directed against the H N and F proteins of chicken A/PMV-t viruses. We have also studied the pathogenicity of the different, pigeon isolates using conventional patho- genicity tests in SPF chickens.

Materials and Methods

V i r u s I so l a t i on

The vi ruses e x a m i n e d in th i s s t u d y were i so la ted f rom af fec ted rac ing p igeons b y inocu la t ion of samples of b r a i n or i n t e s t ine in to t h e a l lan to ic c a v i t y of n ine to e leven day -o ld c m b r y o n a t e d ch icken S P F eggs (VMo-Lohman) . Fo r v i rus i so la t ion a t t e m p t s 10 per cen t w /v suspens ions of in t e s t ines or b r a i n were m a d e in 0.1 ~ p h o s p h a t e buf fe r (PBS) c o n t a i n i n g 100 U penici l l in, 100 ~g of s t r e p t o m y c i n a n d 80 ~g of g e n t a m y e i n pe r ml. Af te r 2 hou r s a t r oom t e m p e r a t u r e , t he s u p e r n a t a n t f luid f rom t h e suspens ions was inocu la t ed in to t he eggs. F ive eggs were used for each sample . The i nocu la t ed eggs were i n c u b a t e d a t 37 ° C a n d cand led daily. The a l lan to ic f luids of dy ing eggs were h a r v e s t e d a n d t e s t ed for h e m a g g l u t i n a t i n g ac t iv i ty .

I so l a t ed v i ruses were ident i f ied as A / P M V - 1 v i ruses b y h e m a g g l u t i n a t i o n i n h i b i t i o n tes t s w i t h reference an t i s e r a p r e p a r e d in r a b b i t s a n d w i t h mouse monoe lona l an t ibodies .

S e ~

Polye lona l reference a n t i s e r u m d i rec ted aga ins t t h e g lycopro te ins of t h e r ep resen ta - t ive ch icken A/PIVtV-I v i rus s t r a ins La Sota a n d I t a l i e n were p r e p a r e d in r abb i t s . The g lyeopro te ins of these v i ruses were solubi l ized us ing oetylglueoside fol lowing t h e m e t h o d of KOHAMA et al. (1981). l~abbi t s were i n j ec t ed s u b c u t a n e o u s l y w i t h a m i x t u r e of 100 ~zg of v i ra l g lyeopro te ins (0.5 ml) a n d comple t e F r e u n d a d j u v a n t (0.5 ml). Th i s i n j ec t i on was r e p e a t e d 7 days la ter . A boos te r i n j ec t ion of 100 ~zg of g lyeopro te ins in P B S was g iven i n t r a v e n o u s l y 3 weeks la te r , t {abb i t s were b led I0 days a f t e r t h e l a s t in jec t ion ,

F o u r d i f fe ren t g roups of monoe lona l an t ibod ie s d i r ec ted aga ins t I-IN were o b t a i n e d p rev ious ly (L~ LONG et al., in p r epa ra t i on ) . A r e p r e s e n t a t i v e monoe lonM of t h e f i rs t t h r e e g roups was u sed in t h i s s t u d y t o g e t h e r w i t h one m o n o e l o n a l d i r ec ted a,gainst t h e F p ro t e in of I t M i e n v i rus . A n t i b o d y 8 C 1 i (group 1) was o b t a i n e d a f t e r i m m u n i z a t i o n of mice w i t h I t a l i e n v i rus whi le an t ibod ie s 4 C3 (group 2) a n d 5 A 1 (group 3) de r ived f rom mice k m e u l a t e d w i t h L a So ta virus .

Serological T e s t s

H e m a g g l u t i n a t i o n i nh ib i t i on tes t s were done in mie rop la t e s us ing d o u b l i n g d i lu t ions of se rum, 1 pe r c en t ch i cken r ed b lood celts a n d four h e m a g g l u t i n a t i n g u n i t s of v i ru s fol lowing t h e m e t h o d of ALLAN a n d Gou~H (1974:). Monoclona l an t ibod ie s were m o u s e asei t ie f lu id w h i c h h a d been d i lu t ed 1/10 in P B S before tes t ing . T i t res were expressed

Characterization of Type 1 Pigeon Paramyxovirus 153

as t h e rec iprocal of t he h ighes t s e r u m d i lu t ion w h i c h caused i n h i b i t i o n of t h e hem- agg lu t ina t ion .

S e roneu t r a l i z a t i on tes t s were done fol lowing t h e s -v i rus n e u t r a l i z a t i o n p rocedu re (BEARD, 1980). Serial t en fo ld d i lu t ions of v i rus were m i x e d w i t h a s t a n d a r d d i lu t ion 1/10 of h e a t i n a c t i v a t e d (56 ° C, 30 minu t e s ) asci tcs fluids. T h e asc i tes -v i rus m i x t u r e s were i n c u b a t e d d u r i n g 2 hou r s a t 37 ° C a n d a s sayed for res idual v i rus b y i n c u b a t i n g s e c o n d a r y cu l tu res of ch i cken e m b r y o f ib rob las t s p r e p a r e d in 24 wells t i t e rp l a t e s . F o r each v i ra l d i lu t ion , 5 wells were i n o c u l a t e d w i t h 0.2 ml of t h e v i rus -asc i tes m i x t u r e .

Contro ls cons i s t ing of m i x t u r e s of v i ra l d i lu t ions w i t h a n e g a t i v e s e r u m were inc luded in each tes t . Af te r 1 h o u r abso rp t ion , cu l tu res were a d d e d w i t h m a i n t e n a n c e m e d i u m , i n c u b a t e d a t 38.5 ° C for 96 hou r s a n d e x a m i n e d for c y t o p a t h i c effects. The n e u t r a l i z i n g i n d e x r ep re sen t s t he difference b e t w e e n t h e v i rus t i t r e s in t h e con t ro l e x p e r i m e n t a n d t he v i rus t i t r e s in t h e m i x t u r e v i rus-asci tes . T h e v i rus t i t r e s were ca l cu la t ed accord ing to REED a n d MUE~C~ (1938).

Thermostability o/the Hemagglutinin

Allan to ic f lu id was cen t r i fuged a t 400 × g for 10 minu t e s . The s u p c r n a t a n t was sea led in to ampou le s a n d f u r t h e r s u b m e r g e d in a w a t e r b a t h a t 56 ° C for se lec ted per iods of t ime a n d t h e n r ap id ly chi l led on ice. The h e m a g g l u t i n a t i o n t i t r e of t h e t r e a t e d v i rus was m e a s u r e d in mic rop la t e s us ing 1 pe r cen t ch icken red b lood cells a n d 50 V1 vo lumes .

Immunopreeipitation and Electrophoretic Analysis o/ Viral Proteins

B a b y H a m s t e r K i d n e y cells ( B H K 21) growing in mono layc r s in 25 cm 2 f lask were in fec ted w i t h t h e d i f fe ren t p igeon isola tes or w i t h t h e I t a ] i en s t r a i n a t t h e mu l t i p l i c i t y of 10 P F U / c e l l in 0.4 ml of M E M - t t e p e s (20 mM; p H 7.4) w i t h 10 pe r cen t foeta l b o v i n e s e r u m for 30 m i n u t e s a t r o o m t e m p e r a t u r e , t h e n i n c u b a t e d a t 37 ° C w i t h 3.6 ml of M E M - H e p e s w i t h 10 per cen t foeta l b o v i n e s e r u m per f lask. A c t i n o m y c i n D (2 tzg/ml) was a d d e d a t 2,5 hou r s pos t - in fec t ion . 35S m e t h i o n i n e - l a b e l i n g was p e r f o r m e d a t 5 hou r s pos t - in fec t ion w i t h 28 ~Ci/ml in 1.8 ml M E M - H e p e s w i t h o u t m e t h i o n i n e s u p p l e m e n t e d w i t h A c t i n o m y c i n e D a n d d ia ]ysed foetal calf se rum. Af te r 1 h o u r ]abel ing, t h e in fec ted cells were w a s h e d twice w i t h T N E (Tris~ttC1 0.01 M, p H 7.4; NaC1 0.15 ~ ; E D T A 0.001 M) a n d lysed w i t h 0.5 ml per f lask of lysis buf fe r ( T N E w i t h 1 pe r cen t T r i t o n X-100) for 10 m i n u t e s a t 4 ° C.

The lysa te was t h e n s p u n in t h e E p p e n d o r f cen t r i fuge for 15 m i n u t e s a t 4 ° C, b r o u g h t to 5 ml w i t h T N E buf fe r m a d e 1 pe r cen t in T r i t o n X- 100, 0.5 pe r cen t in N a D e o x y c h o l a t c a n d 0.1 per cen t SDS ( R I P buffer) a n d clar if ied b y c e n t r i f u g a t i o n in t h e Spinco S W 50.1 ro to r a t 3 6 K for 60 m i n u t e s a t 4 ° C.

R a b b i t a n t i s e r u m or m o n o c l o n a l a n t i b o d y (4 ~1) was m i x e d w i t h 400 ~1 of clar if ied ]ysa te ; t he so]ut ion was i n c u b a t e d for 16 hou r s a t 4 ° C. R a b b i t a n t i - m o u s e Ig s e r u m (4 ~1) was a d d e d a n d a l lowed to r eac t for 1 h o u r a t 4 ° C. I m m u n e complexes were a d s o r b e d on to 50 ~l of a suspens ion of i n a c t i v a t e d Staphylococcus aureus, al lowed to r eac t for 30 m i n u t e s a t 4 ° C.

The bac te r i a l suspens ion was w a s h e d twice in R I P buffer , once in a 1 : 1 R I P / T ~ T E buf fe r a n d once in T N E . T h e y were d i s so lved in 50 ~1 of s ample buf fe r (0.05 M Tris-HC1, p H 6.8 ; 1 pe r cen t SDS ; 1 pe r cen t 2 - m e r c a p t o c t h a n o l ; 10 per cen t v / v glycerol) , h e a t e d a t 100 ° C for 3 m i n u t e s a n d cen t r i fuged to e l imina te S. aureus. T h e s u p e r n a t a n t s were e l ec t rophorezed in 10 pe r cen t SDS-po lyac ry l ami d e gels in d i scon t inous s lab gels b y t h e m e t h o d of LAEMMLI (1970). Af te r e lec t rophores is , t h e gels were fixed, s t a i n e d for f luo rography , d r i ed a n d exposed w i t h K o d a k X-A]~ 5 films.

11"

Tab

le

1. H

emag

g~ut

inat

ion

inh

ibit

ion

and

ser

oneu

tral

izat

ion

test

s w

ith

pig

eon

viru

ses

Vir

use

s

An

tise

ra

La

So

ta

1 2

3 4

5 6

7 8

9 10

11

2,04

8 25

6 25

6 51

2 25

6 25

6 51

2 25

6 64

12

8 1,

024

2,04

8 4,

096

512

512

t,0

24

4,

096

1,02

4 2,

048

1,02

4 1,

024

512

1,02

4 2,

048

640

10,2

40

1,28

0 5,

120

5, t2

0

5,12

0 2,

560

5, t2

0

2,56

0 1,

280

5,12

0 5

,t2

0

5,12

0 0

0 0

0 0

0 0

0 0

>~ 1

,280

~

1,28

0 5,

120

0 0

0 0

0 0

0 0

0 )

1,28

0 ~

1,28

0

La

Sot

a~

Ital

ien

• A

nti

I~

N I

no

no

elo

nM

s a

Gro

up

1

(8C

lt)

Gro

up

2 (

4C3)

G

rou

p 3

(5

A1

) A

nti

F

mo

no

elo

nal

b

1C3

3.25

4.

80

4.55

5.

33

5.33

4.

69

4.54

4.

67

4.88

4.

37

ND

N

D

Vir

use

s

An

tise

ra

12

13

14

15

16

17

18

19

20

21

ItM

ien

© t

La

So

fa a

Ital

ien

a

An

ti }

IN m

on

ocl

on

als

~ G

rou

p

1 (8

Cll

) G

rou

p 2

(4C

3)

Gro

up

3 (

5A

1)

An

ti F

m

onoe

lona

.1 ~

1

C3

512

512

256

512

512

1,02

4 51

2 25

6 12

8 51

2 2,

048

8,19

2 2,

048

1,02

4 1,

024

2,04

8 4,

096

1,02

4 1,

024

1,02

4 51

2 2,

048

10,2

40

2,56

0 2,

560

2,56

0 2,

560

5,12

0 10

,240

5,

120

1,28

0 10

,240

5,

120

0 20

0

0 0

0 0

0 0

0 64

0 0

20

0 0

0 0

20

0 20

0

0

>~4

5.12

5

5.18

5.

49

5.18

4.

87

5.55

4.

12

5.12

4.

34

iFIe

mag

glu

tin

atio

n i

nh

ibit

ion

tes

t b

Ser

on

eutr

aliz

atio

n t

est

Character iza t ion of Type 1 Pigeon P a r a m y x o v i r u s 155

Pathogenicity Tests

In t raeerebra l pa thogen ic i ty index ( ICPI) tests in day-old S P F ehieks and intra- venous pa thogen ic i ty index ( IVPI) test, s in six-week-old S P F chickens were done as descr ibed by ALLA~ et al. (1978).Both tests are es t imated as the t ime t aken for birds to die or to show signs of disease after" infection. I n t he I C P I tes t , birds are scored each day normal (0), sick (1) or dead (2) and a mean score per bi rd per observa t ion is calculated. A m a x i m u m I C P I of 2 means all birds dead wi th in 24 hours and a m i n i m u m of 0 signifies t ha t no signs were recorded dur ing eight days observat ions.

I n the I V P I test , the birds are seored 0 (normal), 1 (sick), 2 (parMysed) and 3 (dead) so t h a t a m a x i m u m index of 3 means all birds dead wi th in 24 hours and a m i n i m u m of 0 represents no clinical signs after 10 days (A:LEXK~Dm~ and PAt~SOI~S, 1984).

Fig. 1. Autorad iograms of SDS-Polyaery lamide gel eleetrophoresis of N D V - I t a l i e n s t ra in and 21 pigeon isolates polypept ides immunopree ip i t a t ed by a R a b b i t an t i N D V - I t a l i e n g lyeoprote in polyelonal serum. The lysates were f rom BI IK-21 cells infected wi th N D V - I t a l i e n s train (I) or wi th 21 pigeon isolates (1 to 21) and label led wi th (ssS)-Methionine. M W = M o l e e u l a r weight pro te in markers : 200,000, 100,000,

92,500, 69,000, 46,000, 30,000

t56 G. MEUI~EMANS et al. :

Results

Serological Tests

I{esu]ts of hemagglutination inhibition and seroneutralization tests using pigeon viruses and chicken paramyxoviruses are given in Table 1.

Fig. 2. Autoradiograms of SDS-Polyaerytamide gel etectrophoresis of BIcfK-21 cell lysates infected with NDV-Italien strain and pigeon isolates, and labelled with (3~S)- Methionine. The Fig. shows differences in molecular weight between ItaIien strain

and pigeon isolates (5--11--14) concerning viral proteins F0, P and !Vl

The hemagglutination of all pigeon viruses was inhibited at high ti tre with polyclonal antisera against Italien and La Sota strains. However, ma, fked differences in titres were observed between La Sota and Italien viruses and the pigeons isolates in tests using antiserum against the glyeo- proteins of La Sota virus. Except for isolates 10, 11 and 17, titres obtained with pigeon viruses were 2 to 5 log2 lower than with La Sota virus.

Monoclonal antibodies 4 C3 of group 2 and 5 A1 of group 3 which are directed against the t IN protein of La Sota virus only inhibited the hem- agglutination of strains I0 and I1 when monoelonal of group 1 which is directed against the HN protein of Italien strain inhibit the hemagglutination of all pigeon isolates at high titre.

Characterization of Type 1 Pigeon Paramyxovirus 157

Monoclonal 1 C 3, directed against the F protein of Italien virus neutral- ized the infectivity of all pigeon isolates, tile neutralizing index being even higher with pigeon than with Italien and La Sofa viruses.

These serological results were confirmed by the immunoprecipitation studies (Figs. 1 and 3).

Imrnunopreeipitation and Electrophoretic Analysis o/ Viral Proteins

Polyclonal anti Italien glycoproteins serum reacts equally well with Italien and pigeon viruses by precipitating HN, F0, P, F1 and M proteins.

Fig. 3. Autoradiograms of SDS-Polyaerylamide gel eleetrophoresis of 21 pigeon isolates polypeptides (1 to 21) immunopreeipitated by an anti-F monoelonM antibody (1C3). R Control lysate of BIIK-21 cells infected with pigeon strain 5 and immunopreeipitated by a Rabbit anti NDV-Italien glyeoproteins polyetonal serum. Mo Control, the same lysate precipitated by a normal Mouse serum. MW Molecular weight protein markers

(see Fig. 1)

158 G. I~InvLES~ANS et al. :

Some q u a n t i t a t i v e differences were however seen be tween isolates; s t ra ins 6,

8 and 9 being less react ive. The i m m u n o p r e e i p i t a t i o n of P a n d M prote ins was unexpec ted and

resul ted f rom con tamina t ion of the oetylglucoside ex t r ac t used for the i m m u n i z a t i o n of the r abb i t s wi th P and M proteins. I t is in teres t ing to note t h a t the molecular weights of F0, P and M prote ins f rom pigeon viruses were di f ferent f rom those of I ta l ien vi rus (Fig. 2); moreover , the molecular

weight of P pro te in va r i ed f rom one pigeon s t ra in to the o ther (Fig. 1). Monoclonal 1 C3 prec ip i t a ted the F prote ins of all pigeon s t rains and

the immunoprec ip i t a t i on tes ts clearly show an absence of the F1 pro te in

for s trMns t0 and 11 (Fig. 3). Prec ip i ta t ion of M prote in of some strains b y the ant i F monoclonal a n t i b o d y (1 C3) was in te rp re ted as non specific as i t occurred also using a control se rum f rom non i m m u n e mouse (Fig. 3, line Mo). The i m m u n o p r e c i p i t a t i o n react ions were less obvious wi th s t rains 6, 8 and 9 as a l r eady observed wi th the polyclonal r abb i t ant iserum. F r o m the sero-

logical and immunopree ip i t a t i on studies, i t m a y be concluded t h a t all pigeon isolates are t rue A/PMV-1 viruses. St ra ins 10 and 11 are different

Table 2. Pathogenecity indexes and thermostability o] the hemagglutinin o/ pigeon A / P . ~ V- l viruses

Date of HA

Strain no isolation Viral titre~ ICPI IVPI stabilit, yb

1 4/83 8.63 1.40 0.43 30 2 8/83 8.38 1.50 0.79 10 3 8/83 8.50 1.50 1.67 10 4 9/83 8.50 1.50 0.26 10 5 9/83 8.32 1.48 0.90 30 6 9/83 8.17 1.38 0.00 60 7 9/83 8.17 1.40 0.05 10 8 9/83 8.38 1.48 0.58 10 9 9/83 8.00 1.40 0.69 30

10 9/83 7.33 0.17 0.00 0 11 9/83 7.50 0.25 0.00 0 t2 10/83 6.50 1.00 0.12 t0 13 10/83 8.50 1.40 0.70 10 14 10/83 8.38 1.20 0.46 30 15 10/83 8.50 t.25 0.17 30 16 10/83 8.32 1.t8 0.00 60 17 10/83 8.68 1.30 0.00 45 18 10/83 8.50 1.38 0.30 45 19 11/83 8.38 1.30 0.00 45 20 11/83 8.50 1.17 0.00 60 21 11/83 7.75 1.28 0.00 10

TCID~0/0.2 ral b In minutes

Characterization of Type t Pigeon Paramyxovirus 159

from the other pigeon isolates and my be classified as lentogenic viruses because their hemagglutination is inhibited by a monoclonal of group 3 which was demonstrated to be specific for lentogenie strains (LE Lo~cG et al., in preparation); moreover, the lack of cleavage of F0 into F1--F2 observed for these strains is also typical of lentogenie viruses (NAGAI et al., 1976).

Pathogenicity Tests

The results of the pathogenicity tests are given in Table 2, they confirm the classification of strains 10 and 11 in the lentogenic group. The other pigeon isolates tested gave a mean ICPI of 1.34, range t.00 to 1.50 and a mean IVPI of 0.37, range 0 to 1.67. I t is interesting to note that whilst the ICPI values fall into a tight group about the mean, the IPVI values show much greater variation, isolates with equally high ICPI having quite different IVPI. This heterogenicity in pathogenicity denotes biological variations between isolates made during a relative short period of time, it is also reflected in the differences observed in the thermostability of the hemagglutinin of the different viruses.

Discussion

Viruses isolated from clinically sick pigeons in 12 different countries were recently demonstrated to be antigenieally indistinguishable (ALExA~- DEE et al., 1985), they belong to the A/PMV-1 serotype but, show some variation from classical strains (BIANCIFIORI and FIORI~I, i983; I~ICHTER, 1983; VIAE~E et al., i983).

Our results proved unequivocally pigeon viruses to be A/PMV-1 viruses having a closer antigenic relationship with the Italien virus of chickens than with the La Sota virus. Using polyelonal and monoelonal antisera against the Italien virus, we were indeed unable to make a precise difference between chicken Italien and pigeon viruses by hemagglutination inhibition and seroneutralization tests. However, SDS-PAGE showed an augmentation of the M.W. of F0 and a decrease of the M.W. of M proteins when pigeon viruses were compared to Italien virus; these variations could be used for differentiation of pigeon viruses from chickens A/PMV-I viruses.

ALEXANDER et al. (1984) reported that the hemagglutination activity of pigeon isolates was not inhibited by the monoelonal antibodies directed to the HNI epitope of Ulster 2 C which allowed a rapid test for distinguishing between the pigeon isolates and the vast majori ty of A/PMV-1 viruses (R~TssELL and ALEXANDER, 1983). Similar results were obtained in our s tudy using monoclonal 4 C3 (group 2) which is directed against the t-IN protein of the La Sofa strain.

Both observations and the fact that monoclonals directed against the H N and F proteins of the chicken Italien virus detected all pigeon isolates

160 G. ~/~EULEIvIANS et a l . :

give some indication that pigeon isolates probably derive from chicken velogenie strains.

Marked differences in virulence were recorded among pigeon isolates. Serological results and pathogenicity testing clearly demonstrate the presence of two lentogenic strains (10 and 11) similar to vaccinal A/PMV-1 viruses. The isolation of lentogenic A/PMV-t viruses from pigeons showing respiratory disease has already been reported in absence of epizootics in pigeons (VIND~VOGEL et al., 1982).

All the other strains may be classified as pathogenic neurotropie viruses. The marked variations in IVPI and the rather similar ICPI values recorded for these strains is in agreement with the results obtained by AnEXA~DER. and PAI~S0~'S, 1984; AL~:XAh'DE~ et al., 1984b; 1984c. The variations in virulence between pathogenic isolates as the differences observed in the thermostability of their hemagglutinin could result from selection of different viral subpopulations during passages in pigeons.

The existence of velogenic isolates among pigeon viruses such as strain 3 represents a possible source of infection for commercial poultry flocks. Oecurenee of Newcastle disease in poultry in Great Britain by contamination of stored food with pigeon faeces has recently been reported (A~o~YM:0us, 1984). Eradication of A/PMV-1 infection in pigeons would therefore be of great importance and could be achieved by obligatory vaccination of all racing pigeons using inactivated A/PMV-1 vaccines normally used in chickens.

References

1. ANONymOUS : Newcastle disease: Outbreaks linked to pigeons. Vet. Bee. 114, 305-- 306 (1984).

2. ALEXANDE:a, D. J., P~RSONS, G. : Avian paramyxovirus t~ype 1 infections of racing pigeons: 2 Pathogenicity experiments in pigeons and chickens. Vet. R.ee. 114, 446--449 (1984).

3. ALEXANDEI¢, D. J., P~USSELL, P. H., COLLINS, 1~. S.: Paramyxovirus type 1 infections of racing pigeons: 1. Charaeterisation of isolated virus. Vet. lace. 114, 444- ~o446 (1984a).

4. ALEX~tNDER, D. J., WILSON, G. W. C., THAIN, J. A., LIS~E~, S. A. : Avian paramyxo- virus type 1 infection of racing pigeons: 3 Epizootiologieal considerations. Vet. t~ee. 115, 213--216 (1984b).

5. ALEXANDER, D. J., RVSSEL~, P. H., PA~SO:~S, G., ABU ELZEIN, E. M. E., BalmOU~, A., CEI~NIK, K., ENGSTB, O~I, B., FEVEREIt~O, M., FLEURY, H. A. J., GUITT:ET, ]V[., KALET~, E. F., KIHI~, U., KOSTEaS, J., LOMNICZI, B., MmST]~, J., MEULES~ANS, G., NEII.O~IE, K., PETEK, ~i., POKONUNSKI, S., POLTEN, B., PI~IP, M., R, ICHTER, R., S~-GI~Y, E., S_~-SiBERG, Y., SPANO~HE, L., TUSIOVA, B. : Antigenic and biological characterization of avian paramyxovirus type i isolates from pigeons. An inter- national collaborative study. Avian Path. 14, 365--376 (1985).

6. ALLAN, W. H., Gou~II, R. E.: A standard hemagglutinabion inhibition test for Newcastle disease. I. A comparison of macro and micro methods. Vet. Rec. 95, t2O--t 23 (1974).

Characterization of Type 1 Pigeon Paramyxovirus 161

7. ALLAN, W. m., LA:~'CASTEI~, J. E., TOTH, B. : Newcastle disease vaccines. Food and Agriculture Organization of the United Nations. Rome, 74 (1978).

8. BEARD, C. W.: Serologic procedures. In : I-IITCH~ER, S. B., PUrChASE, H. G., ~¥I~LIA~S, G. A. (eds.), Isolation and Identification of Avian Pathogens. 2nd ed., The Association of American Avian Pathologists t29--135. New York: Creative Print ing Co. 1980.

9. BIANOIFIORI, F., :FIO~I~I, A.: An occurrence of Newcastle disease in pigeons: Virological and serological studies on the isolates. Comp. Immun. Microb. and Infect. Dis. 6, 247--..252 (1983).

10. EIZA, M., 0~v~E~, E. A. : A natural outbreak of Newcastle disease in pigeons in the Sudan. Vet. Rec. 114, 297 (1983).

11. GUITTET, ~J[., BENNEJE&I% G.: La paramyxovirose du pigeon. L'aviculteur 445, 59--62 (1984).

12. KOHA~A, T., GA~'rEN, W., KL~NK, H. D.: Changes in conformation and charges paralleling proteolytic activation of Newcastle disease virus glycoproteins. Virol. 111, 364--376 (1981).

13. LAE~MLI, V. K. : Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 277, 680--685 (1970).

14. LE LONG., PO~TETELLE, D., GIIYSDAEL, J., GONZE, ~[., BU:aNY, A., MEULEFZANS, G. : Monoclonal antibodies to hemagglutinin and fusion glycoproteins of Newcastle disease virus. (In preparation).

15. N~GAI, Y., K:LENK, i. D., RoT% R. : Proteolytic cleavage of the viral glyeoproteins and its significance for the virulence of Newcastle disease virus. Virology 72, 494-- 508 (1976).

16. PERIIVI, S., MA~AS'rONI, G., PASCUCCl, S.: Sull'attuale epizooia di malattia di Newcastle nei piccioni. Selezione Vet. 23, 129--130 (1982).

17. RICHTER, R.: III. Tagung der Faehgruppe Geflfigelkrankheiten. Thema: Vogel- krankheiten. Schwerpunkt Taube. Deutsche Veterin~rmedizinische Gesellschaft, 86--95 (1983).

18. I~,EED, L. J., ~/[UENGH, H. : A simple method for estimating fifty per cent endpoints. Am. J. Hyg. 27, 493---497 (1938).

19. RUSSELL, P. H., ALEXANDER, D. J.: Antigenic variation of Newcastle disease virus strains detected by monoelonal antibodies. Arch. Virol. 75, 243--245 (I 983).

20. %rI_&EN-E, N., SPANOGHE, L., DEVRIESE, a., BI.INENS, B., DEVOS, A. : Paramyxovirus bij duiven. Vlaams Dierg. Tijdschr. 52, 278--286 (1983).

21. VINDEVOGEL, I~., THIR¥, E., PASTOEET, P., MEULEMANS, ~.: Lentogenic strains of Newcastle disease virus in pigeons. Vet. Ree. 114, 305~--306 (1982).

Authors' address : Dr. G. IVIEULEMANS, National Institute for Veterinary Research, Groeselenberg 99, B-1180 Brussels, Belgium.

Received April 17, 1985