Veterinary Microbiology, 22 (1990) 171-178 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

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Isolation and C h a r a c t e r i z a t i o n of A t t e n u a t e d P l a q u e Variants of Infectious Bursal D i s e a s e Virus

K. SAIJO 1, M. HIGASHIHARA1., Y. FUJISAKI 1and M. MATUMOTO 2

1Research Center for Veterinary Science, The Kitasato Institute, Kashiwa-shi, Chiba 277 (Japan) 2Department of Virology, The Kitasato Institute, Minato-ku, Tokyo 108 (Japan) (Accepted 17 October 1989)

ABSTRACT Saijo, K., Higashihara, M., Fujisaki, Y. and Matumoto, M., 1990. Isolation and characterization of attenuated plaque variants of infectious bursal disease virus. Vet. Microbiol., 22: 171-178. Attenuated plaque variants were obtained from infectious bursal disease virus adapted to chick embryo cell cultures. The large plaque (Lp) clone and the small plaque (Sp) clone formed homogeneous plaques about 5 and 1 mm in diameter, respectively. Neutralization tests indicated that these clones differed little from their parent strain in antigenicity. Sp clones showed a retarded growth rate in chick embryo cell cultures as compared with Lp clones. The clones were significantly less pathogenic for chick embryos than the parent strain, although Lp clones were more pathogenic than Sp clones, and they were much less pathogenic for 1-day-old chicks and 28day-old chickens. Both clones had immunizing potency in 28-day-old chickens, although the Lp clone had a somewhat higher potency than the Sp clone. These findings suggest the Lp and Sp clones, in particular the Lp clones, to be useful as live virus vaccine strains.

INTRODUCTION

Infectious bursal disease (IBD) is of considerable economic interest for the poultry industry. It results in a complete destruction of the bursa of Fabricius (BF), the central organ of the B-cell immune system of birds. IBD virus is a member of the genus Birnavirus (Murphy, 1988). The present report describes the establishment and characterization of attenuated plaque variants derived from IBD virus adapted to chick embryo cell cultures. *To whom correspondence should be addressed.

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MATERIALS AND METHODS

Chicken embryos and chickens These were obtained from fertile eggs of specific pathogen-free flocks maintained at Yakumo Farm, Kitasato University, Hokkaido, Japan. The flocks were monitored periodically by serological and clinical tests and confirmed to be free from common bacterial and viral pathogens including IBD virus.

Chick embryo fibroblast (CEF) cultures Cells were prepared from 10-day-old embryos and grown in Eagle's minimum essential medium (MEM) containing 5% calf serum. The content of calf serum was reduced to 2% for maintenance medium. V/ruB

The RF-1 strain of IBD virus was isolated in 1972 from the homogenate of the BF of an infected chicken {Izawa et al., 1978), and used in the present study after 3 passages in chick embryos (RF-lwt) and after 25 passages in chick embryos followed by 15 passages in CEF cultures (RF-ltc: Nagabayashi et al., 1974). The RF-lwt virus was grown in 28-day-old chickens. The birds were orally inoculated with 103 50% egg infective doses (EIDso) and sacrificed 3 days later. Their BFs were homogenized to make a 20% suspension in PBS (0.15 M NaC1, 0.01 M phosphate buffer, pH 7.5 ) and clarified by centrifugation. RF-ltc virus was grown in CEF cultures prepared in 150-cm2 plastic flasks (Corning, U.S.A.). The cultures were inoculated with 0.1 to 0.5 50% tissue culture infective doses (TCIDso) per cell and incubated at 37 °C for 2 to 3 days. The cultures were disrupted by 3 cycles of freezing and thawing and clarified by centrifugation. Virus materials of the Lp and Sp clones (see below) obtained from RF-ltc were prepared in CEF cultures in the same manner as the RF-ltc virus. These virus materials were stored at - 8 0 ° C until use.

Plaque formation Virus was inoculated in 0.1 ml volumes into CEF monolayer cultures prepared in 60-mm plates (Corning, U.S.A. ). After adsorption at 37 ° C for 60 rain, the cultures were overlayed with MEM containing 5% calf serum and 1% Noble agar (Difco, U.S.A.). The cultures were incubated at 37°C for 4 days and stained with 0.02% neutral red. The infectivity was expressed in plaque forming units (PFU).

Infectivity assay in chick embryos Serial tenfold dilutions of the RF-lwt strain were inoculated in 0.1 ml volumes onto the chorio-allantoic membrane of ll-day-old embryonated eggs employing 5 eggs per dilution. The inoculated eggs were further incubated at 37 °C

ATTENUATED PLAQUE VARIANTS OF INFECTIOUS BURSAL DISEASE VIRUS

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for 7 days, embryo deaths and/or specific IBD lesions were counted and the EIDso was calculated by the method of K~irber. Infectivity assay in CEF cultures Cell cultures were prepared in 24-well semi-microplates (Corning, U.S.A.). Serial tenfold dilutions of the virus material were inoculated in 0.1 ml volumes into cell cultures employing 5 wells per dilution. The inoculated cultures were incubated at 37°C for 6 days, examined for any cytophatic effect (CPE) and the TCID~o was calculated by the method of K~irber. Antisera Antisera against Lp, Sp, RF-ltc and RF-lwt viruses were prepared in 28day-old chickens, employing 10 birds for each virus. The birds were orally inoculated with 105 TCIDs0 of Lp and Sp and RF-ltc viruses and with 1017 EIDso of RF-lwt virus. Sera were obtained 28 days later and pooled. Neutralization (NT) test The NT test was carried out by the plaque reduction method. Serial twofold dilutions of the serum inactivated for 30 min at 56 ° C were mixed with equal volumes of maintenance medium containing 200 PFU of virus per 0.1 ml and incubated for 60 min at 37°C. The virus-serum mixtures were inoculated in 0.1 ml volumes into CEF monolayers prepared in 60-mm plates. After absorption for 60 min at 37°C, the cultures were covered with overlay medium, incubated for 4 days at 37°C and stained with neutral red. The NT titer was expressed as the reciprocal of the highest serum dilution showing 50% or greater reduction. In some experiments, NT tests were performed by the inhibition of CPE in microplates. Serial twofold dilutions of the serum inactivated for 30 min at 56 °C were made in 0.025 ml volumes with maintenance medium in transfer plates employing 4 wells for each serum dilution and each serum dilution was mixed with an equal volume containing 100 TCIDso per 0.025 ml of virus. The virus-serum mixtures were incubated for 30 min at 37 ° C, then transferred into wells of flat-bottomed microplates (Corning, U.S.A.) containing CEF cultures. After incubation for 6 days at 37°C, the cultures were examined for any CPE and the antibody titer was expressed as the reciprocal of the serum dilution showing 50% CPE inhibition calculated by the method of K~irber. NT titers of 10 or higher were taken as positive. RESULTS

The RF-ltc virus was found to produce circular clear plaques with sizes ranging from 1 to 5 mm in diameter in CEF cultures. Plaque cloning repeated 3 times resulted in isolation of large and small plaque clones. The large plaques

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(Lp) and small plaques (Sp) were homogeneous in size, about 5 and 1 mm in diameter, respectively. The clones showed no changes in the plaque size after 10 serial passages in CEF cultures. The Lp and Sp clones and RF-ltc strain were serologically compared by the plaque reduction N T test. The results are summarized in Table 1. Antisera against Lp, Sp, RF-ltc and R F - l w t viruses showed little differences in the NT titer between Lp, Sp and RF-ltc viruses, indicating that Lp and Sp clones differed antigenicity little from their parent strain RF-ltc. The multiplication of Lp and Sp clones in CEF cultures was compared. CEF cultures were prepared in 25 cm 2 plastic flasks, inoculated with 0.2 TCIDso per cell of virus and incubated at 37 ° C. At intervals two flasks were taken from each group of cultures and pooled culture fluids were assayed for extracellular virus. The same volume of maintenance medium was added to the cell sheet after two washings with PBS. The cells were disrupted by 3 cycles of freezing and thawing and used to assay intracellular virus. The results are illustrated in Fig. 1. The Lp clone replicated more rapidly than the Sp clone. The intraTABLE1 Neutralization (NT) test of Lp and Sp clones and RF- ltc strain of IBD virus Antiserum

NT titer in log of antiserum against virus

Lp clone Sp clone RF-ltc RF-lwt

Lp clone

Sp clone

RF-ltc

4.10 3.71 4.01 3.84

4.01 3.88 4.01 3.90

4.17 4.01 4.21 4.23

"'o--o--o--o--o . . . . . . . . . .

.............

6 s

e . . e . . e ,Q



'.7.,

4 .=. > o

_ . Lp clone

0----0 o.... o

Extracellular Intracellular

~/ /

^ ~p

e---.-e

Extrace

virus virus

3

-0

v-

.o,;~,; ~,'~"/~=,,

clone

• .... •

lular virus

Intracellular

virus

2 I

0

i

4

I

I

8

I

I

12

I

I

16

I

I

20

Hours

I

I

I

24

48

of incubation

Fig. 1. Multiplication of Lp and Sp clones from the IBD virus in CEF cultures.

ATTENUATED PLAQUEVARIANTSOF INFECTIOUS BURSALDISEASE VIRUS

175

cellular titer of the Lp clone reached a plateau after 14 h. Thereafter the extracellular titer increased slightly. The plateau of the Lp clone was about one loglo unit higher than that of the Sp clone. The pathogenicity for chick embryos was compared between Lp, Sp, RF-ltc and R F - l w t viruses. Eleven-day-old embryonated eggs were inoculated on the chorio-allantoic membrane with 104 TCIDso of Lp, Sp and RF-ltc viruses and with 1017 EIDso of R F - l w t virus, employing 25 eggs for each virus. The inoculated eggs were incubated at 37 °C for 7 days. The Lp clone killed 21 of the 25 embryos (84%) within 7 days of observation, but the Sp clone killed no embryos. On the other hand embryonated eggs inoculated with RF-lwt and RFltc viruses showed 100 and 92% embryo death, respectively (Table 2). The embryos were examined macroscopically. The Lp clone induced congestion of the liver and swelling of the spleen in all the 25 embryos, small necrotic foci of the liver and spleen in 9 of the embryos and subcutaneous congestion and petechial hemorrhages in 17 of the embryos. On the other hand, the Sp clone produced congestion of the liver and swelling of the spleen in all the inoculated embryos, but no necrosis of the liver and spleen. RF-ltc virus produced macroscopic lesions quite similar to those of Lp clone, whereas the RFlwt virus produced more severe macroscopic lesions in all the inoculated embryos. The pathogenicity for 28-day-old chickens was compared between Lp, Sp, RF-ltc and R F - l w t viruses. Chickens were inoculated orally with 10 ~ TCID~o of Lp and Sp clones and the RF-ltc strain and with 1017 EIDso of the RF-lwt strain. The R F - l w t strain induced typical symptoms of IBD, whereas the other viruses produced no clinical signs of illness. Four days after inoculation the BF was removed, weighed and examined macroscopically. Lp and Sp clones induced localized necrosis in 4 and 1 of the 10 birds, respectively. On the other hand RF-ltc virus induced necrosis in 9 of the 10 birds, but RF-lwt induced necrosis in all the inoculated birds. Birds inoculated with RF-lwt virus showed TABLE 2 Pathogenicity for chicken embryos of Lp a n d Sp clones and R F - l t c and R F - l w t strains of IBD virus Virus

Lp clone Sp clone RF-ltc RF-lwt

Number of eggs inoculated

Cumulative embryo deaths 2~

3

4

5

6

7

25 25 25 25

0 0 0 0

0 0 5 0

5 0 9 11

8 0 15 15

20 0 22 17

21 0 23 25

"Days after inoculation.

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swollen yellowish and gelatinous bursas in many cases (Table 3 ). No significant difference was observed in the mean bursa/bodyweight ratio in the chicken groups. Their bursal tissues were histologically examined. The lesions in the lymph follicle and interstitium of BF were graded and scores 0, 1, 2 and 3 were given to no, slight, intermediate and severe lesions, respectively. The lesion index was calculated by dividing the total score by the number of inoculated chickens and used for comparison of the severity of pathological changes. The results indicated that Lp and Sp clones induced only slight lesions when compared with those of RF-ltc virus. Lp clone induced somewhat more severe lesions than the Sp clone except for interstitial edema. The pathogenicities of the Lp, Sp, RF-ltc and R F - l w t virus were compared for 1-day-old chicks. Chicks were inoculated in the same manner as in 28-dayold chickens. Chicks inoculated with Lp, Sp and RF-ltc viruses showed no clinical signs of illness. Chicks inoculated with RF-lwt virus showed a reduced bodyweight gain, but no typical signs of IBD. Five weeks after inoculation the birds were autopsied and their BF was removed, weighed and macroscopically examined. RF-lwt virus induced atrophic and yellowish bursas in all the chicks, but no necrosis in any of the inoculated chicks. RF-ltc virus induced atrophic and yellowish bursas in some cases, and Lp and Sp clones induced slight lesions when compared with those of RF-ltc virus (Table 4). The mean ratio of bursa to bodyweight was significantly reduced in chicks inoculated with RF-ltc and R F - l w t viruses, but not in chicks inoculated with Lp and Sp clones. Various amounts of virus, 102 to 10~ TCIDso, were orally inoculated into 28day-old chickens and 14 days later the birds were orally challenged with 1017 EIDso of R F - l w t virus. All the chickens immunized with 104 TCIDso or more of Lp clone and with 105 TCIDso or more of Sp clone resisted the challenge (Table 5 ). N T titers against the virus were determined by the CPE inhibition method at the time of challenge. All the chickens resisting challenge had posTABLE3 Pathogenicity for 28-day-old chickens of Lp a n d Sp clones a n d RF- l t c a n d RF- l w t strains of IBD virus Virus

Lp clone Sp clone RF-ltc RF-lwt

Changes in the bursa of Fabricius

Number of birds inoculated

N u m b e r of birds showing clinical symptoms

Edematous and swollen

Yellowish

Gelatinous

Necrotic

10 10 10 10

0 0 0 10

0 0 0 6

0 0 0 8

0 0 0 7

4 1 9 10

ATTENUATEDPLAQUEVARIANTSOF INFECTIOUSBURSALDISEASEVIRUS

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TABLE 4 Pathogenicity for 1-day-old chicks of Lp and Sp clones and RF-ltc and RF-lwt strains of IBD virus Virus

Lp clone Sp clone RF-ltc RF-lwt

Number of birds inoculated

Number of birds showing clinical symptoms

Changes in the bursa of Fabricius Edematous and swollen

Yellowish

Necrotic

5 5 5 5

0 0 0 5"

1 2 3 5

0 0 2 5

0 0 0 0

"Reduction of bodyweight gain. TABLE 5 NT antibody production and resistance to challenge with RF-lwt IBD virus of 28-day-old chickens immunized with varying doses of Lp and Sp clones of the virus Virus

Virus dose"

Number of birds inoculated

Number of NT antibody positive birds b

Number of birds resisting challengec

Lp clone

l0 s 105 104 103 102

10 10 10 10 10

10 10 10 6 0

10 10 10 6 0

Sp clone

106 10~ 104 103 102

10 10 10 10 10

10 10 4 0 0

10 10 4 0 0

"TCIDso/bird. bNT antibody titers were determined by the CPE inhibition method. cChallenged with oral dose of 10 ~7 EIDso of RF-lwt virus. i t i v e N T t i t e r s , f r o m 14 t o 2560, w h e r e a s i n t h e b i r d s s u c c u m b i n g t o c h a l l e n g e t i t e r s w e r e i n v a r i a b l y l e s s t h a n 10. T e n 1 - d a y - o l d c h i c k s w e r e i n o c u l a t e d s u b c u t a n e o u s l y w i t h 5 X 104 T C I D s o o f t h e L p c l o n e o r w i t h 5 × 105 T C I D ~ o o f t h e S p c l o n e . T h e i n o c u l a t e d c h i c k s developed no clinical abnormalities in the 5 weeks of observation. Ten 1-day-

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old chicks were orally inoculated with 104 TCIDso of the Lp clone or with l0 s TCIDso of the Sp clone. The inoculated chicks developed N T titers of 320 or higher in 3 weeks, when tested by the plaque reduction method. DISCUSSION Izawa et al. (1978) reported the attenuation of IBD virus whereas Cursiefen et al. (1979) and Lange et al. (1987) have reported the attenuation and the plaque formation of IBD virus. In the present study attenuated plaque variants were obtained from the RF-1 strain of IBD virus adapted to CEF cultures. Lp and Sp clones formed homogeneous plaques about 5 and 1 m m in diameter, respectively. N T tests indicated t h a t Lp and Sp clones differed little from their parent strain in antigenicity. The Sp clone showed a retarded growth rate in CEF cultures as compared with the Lp clone. Lp and Sp clones were significantly less pathogenic for chick embryos t h a n the parent strain, although the Lp clone was more pathogenic t h a n the Sp clone, and they were much less pathogenic for 1-day-old chicks and 28-day-old chickens. Both clones had immunizing potency in 28-day-old chickens, although the Lp clone had a somewhat higher potency t h a n the Sp clone. These findings suggest the Lp and Sp clones, in particular the Lp clone, to be useful as live virus vaccine strains.

REFERENCES Cursiefen, D., K~iufer,I. and Becht, H., 1979. Loss of virulence in a small plaque mutant of the infectiousbursal disease virus. Arch. Virol., 59: 39-46. Izawa, H., Eiguchi,Y. and Nagabayashi,T., 1978. Attenuation of infectiousbursal disease virus by serial passage through chicken embryonatedeggs and chicken and duck embryonicfibroblasts. Virus, 28:41-45 (in Japanese). Lange, H., Mfiller,H., K~iufer,I. and Becht, H., 1987. Pathogenicand structural properties of wild type infectious bursal disease virus (IBDV) and virus growth in vitro. Arch. Virol., 92: 187196. Murphy, F.A., 1988. Virus taxonomy and nomenclature. In: E.H. Lennet, P. Halonen and F.A. Murphy (Editors), Laboratory Diagnosisof InfectiousDiseases, Vol. 2, Springer-Verlag, New York, pp. 153-176. Nagabayashi, T., Izawa, H. and Soekawa, M., 1974. The occurrence of cytophatic effect of an isolate of infectiousbursal diseasevirus. Kitasato Arch. Exp. Med., 47: 57-61.

Isolation and characterization of attenuated plaque variants of infectious bursal disease virus.

Attenuated plaque variants were obtained from infectious bursal disease virus adapted to chick embryo cell cultures. The large plaque (Lp) clone and t...
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