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Reversion of molecularly engineered, partiallyattenuated, very virulent infectious bursal diseasevirus during infection of commercial chickensR. Raue a , M. R. Islam b , M. N. Islam b , K. M. Islam b , S. C. Badhy b , P. M. Das b & H.Müller aa Institute for Virology, Faculty of Veterinary Medicine , University of Leipzig , D-04103,Leipzig, Germanyb Department of Pathology, Faculty of Veterinary Science , Bangladesh AgriculturalUniversity , Mymensingh, 2202, BangladeshPublished online: 19 Oct 2010.

To cite this article: R. Raue , M. R. Islam , M. N. Islam , K. M. Islam , S. C. Badhy , P. M. Das & H. Müller (2004) Reversionof molecularly engineered, partially attenuated, very virulent infectious bursal disease virus during infection ofcommercial chickens, Avian Pathology, 33:2, 181-189

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Reversion of molecularly engineered, partiallyattenuated, very virulent infectious bursal diseasevirus during infection of commercial chickens

R. Raue1, M. R. Islam2, M. N. Islam2, K. M. Islam2, S. C. Badhy2,P. M. Das2 and H. Muller1*

1Institute for Virology, Faculty of Veterinary Medicine, University of Leipzig, D-04103 Leipzig, Germany and2Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh2202, Bangladesh

A molecularly cloned, tissue culture-adapted infectious bursal disease virus (BD-3tc) was generated from a veryvirulent strain by the reverse genetics approach following site-directed mutagenesis (Q253H and A284T inVP2). The pathogenicity of BD-3tc was tested in commercial chickens. The wild-type strain (BD-3wt) and themolecularly cloned parental strain (BD-3mc) were included for comparison. The subclinical course of thedisease, with delayed and milder pathological lesions followed by quick follicular regeneration in the bursa ofFabricius in BD-3tc-inoculated birds, suggested that these amino acid substitutions made BD-3tc partiallyattenuated. However, severe bursa atrophy was observed at 14 days after inoculation. Reverse transcription-polymerase chain reaction coupled with restriction enzyme analysis revealed that both point mutations in BD-3tc had reverted 14 days after inoculation. Further investigations demonstrated that the codon for amino acid atposition 284 had already reverted to the wild-type phenotype (T284A) 3 days after inoculation.

Introduction

Infectious bursal disease virus (IBDV) is a double-stranded RNA virus belonging to the genusAvibirnavirus in the family Birnaviridae (Dobos et

al. , 1979; Leong et al ., 2000). The genome consistsof two segments of double-stranded RNA (Mulleret al. , 1979). The major open reading frame in thelarger genome segment A encodes a polyproteinthat is co-translationally and autocatalyticallycleaved into the two structural viral proteins VP2and VP3, and a viral protease VP4 (Muller &Becht, 1982; Azad et al. , 1985; Hudson et al. ,1986). A second partially overlapping open readingframe in segment A encodes a non-structuralprotein, VP5 (Mundt et al. , 1995), which hasbeen suggested to be a transmembrane proteininvolved in the release of virus from infected cells(Lombardo et al. , 2000; Yao & Vakharia, 2001).

The smaller segment B encodes the multifunctionalprotein VP1 predicted to have RNA-dependentRNA polymerase (Spies et al. , 1987) and cappingenzyme (Spies & Muller, 1990) activities.

There are two serotypes of IBDV (McFerran etal. , 1980). Serotype 1 causes a highly contagiousdisease known as infectious bursal disease (IBD),or Gumboro disease, in chickens 3 to 6 weeks ofage. It also produces profound immunosuppressionby the destruction of growing B lymphocytes in thebursa of Fabricius. In the classical form of thedisease, the mortality rates range from 1% to morethan 50% in specific pathogen free (SPF) chickensdepending on the viral dose inoculated (van denBerg et al. , 2004). Layer chickens are moresusceptible than broilers. In pullet flocks depres-sion in egg production and deterioration in eggshelland internal quality of eggs are also observed. Bothbroiler and pullet flocks may become refractory to

*To whom correspondence should be addressed. Tel: �/49 341 9738200. Fax: �/49 341 9738219. E-mail: virology@vetmed.uni-leipzig.de

Received 19 August 2003. Provisionally accepted 13 October 2003. Accepted 27 October 2003

Avian Pathology (April 2004) 33(2), 181�/189

ISSN 0307-9457 (print)/ISSN 1465-3338 (online)/04/20181-09 # 2004 Houghton Trust LtdDOI: 10.1080/03079450310001652112

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live attenuated vaccines against respiratory diseasessuch as infectious bronchitis (Pejkovski et al. , 1979)and Newcastle disease (Higashihara et al. , 1991).In broilers, immunosuppression is denoted by ahigh prevalence of viral respiratory infections andelevated mortality due to airsacculitis and colisep-ticemia during the terminal one-third of the 6 to 8week growing cycle.

Since 1986, very virulent (vv) strains of IBDVhave emerged in Europe, which can cause up to70% flock mortality in laying pullets and up to100% in SPF chickens (Chettle et al. , 1989; van denBerg et al. , 1991, 2004). These strains can causelesions typical of IBDV and are antigenicallysimilar to the classical strains (Eterradossi et al. ,1992). Remarkably, however, vvIBDV can establishinfection in the face of maternally derived anti-bodies as well as antibody levels after vaccinationthat were previously protective against classicalstrains (Hassan et al. , 2002). Meanwhile vvIBDVinfections also have been observed in Africa, Asia(for example, Eterradossi et al. , 1999; Zierenberg etal. , 2000, 2001; Parede et al. , 2003; van den Berg etal. , 2004) and in South America (Ikuta et al. ,2001), but not in Australia (Sapats & Ignjatovic,2002). Some of the recent advances in the under-standing of the structure, morphogenesis andmolecular biology of the virus as well as in thedevelopment of new diagnostic approaches andnew strategies for vaccination against IBD havebeen reviewed recently (Lukert & Saif, 1997; vanden Berg, 2000, Muller et al. , 2003).

Wild-type IBDV, particularly vvIBDV, normallydoes not grow in cell culture. Yamaguchi et al .(1996a) adapted two vvIBDV strains to replicationin chicken embryo fibroblast (CEF) cell culture byrepeated passages in chicken embryos and CEF cellculture, which resulted in marked attenuation. Itwas suggested that amino acid substitutions atpositions 279 and 284 of the capsid protein VP2could be responsible for attenuation of the tissueculture adapted strains (Yamaguchi et al. , 1996b).Subsequently, Lim et al. (1999) rescued a mutant ofvvIBDV strain, which grew in CEF cell culture, bythe reverse genetics approach after introducingthese two amino acids substitutions by site-directedmutagenesis. However, Mundt (1999) and vanLoon et al. (2002) demonstrated that amino acidexchanges at positions 253 and 284 resulted in

tissue culture adaptation of a chimeric IBDV and avvIBDV (UK661), respectively. These mutationsresulted in the attenuation of UK661 for SPFchickens. In the present paper we report on theadaptation of another vvIBDV strain, BD 3/99(Islam et al ., 2001a), to CEF cell culture by aminoacid substitutions at positions 253 and 284 of theVP2. We show that the rescued tissue culture-adapted strain (BD-3tc) is partially attenuated forcommercial chickens, and also demonstrate thatthe mutations revert during replication in the bursaof Fabricius of infected chickens.

Materials and methods

Full-length cDNA clones of vvIBDV strain BD 3/99

Establishment of the complete nucleotide sequences and construction of

full-length clones of the both genome segments A and B of a

Bangladeshi vvIBDV (BD 3/99) have been described (Islam et al .,

2001b). The sequence data have been submitted to GenBank (accession

numbers AF352776 and AF36270, for segment A and segment B,

respectively).

Site-directed mutagenesis and rescue of the mutant virus

A megaprimer polymerase chain reaction (PCR) method (Barik, 1997)

was used for site-directed mutagenesis of nucleotides to introduce

amino acid substitutions at positions 253 (Q0/H) and 284 (A0/T) of

the VP2. The two mutations were introduced successively in two

separate events. The primers used are presented in Table 1.

First, the INCO-DC Primer #3 and the Mutagenic Primer Q253H

were used in a PCR for the synthesis of a megaprimer having the desired

mutation. A plasmid containing the full-length BD 3/99 cDNA was

used as the template. Then the synthesized megaprimer and the INCO-

DC Primer #4 were used in another PCR on the same template for the

amplification of a 677 base pair (bp) cDNA fragment containing the

desired mutation. The product was digested with restriction enzymes

Bsu 15I and Spe I, and the resulting 349 bp long fragment was inserted

into the original full-length clone replacing the corresponding region.

The same strategy was used to introduce the second mutation, except

that the Mutagenic Primer A284T was used and the BD 3/99 cDNA

containing the first mutation served as the template in the PCR. The

expected mutations were confirmed by appropriate restriction enzyme

analysis.

The procedure for regenerating and rescuing infectious virus particles

from the cDNA, adopted from Mundt & Vakharia (1996), has been

described in detail (Islam et al ., 2001b). Briefly, in vitro synthesized

capped RNA transcripts of the BD 3/99 cDNA having the two

mutations were transfected in CEF cells in the presence of a liposome

(Lipofectin; Invitrogen, Karlsruhe, Germany). The expression of viral

proteins in transfected cells was confirmed 24 h later by indirect

immunofluorescence assay using polyclonal rabbit-anti-IBDV serum.

At 96 h post transfection, the supernatant of the transfected cells was

passaged in fresh CEF cell monolayers. After manifestation of the

Table 1. Primers used for site-directed mutagenesis and RT-PCR

Primer Sequencea Nucleotide positionb Orientation Reference

INCO-DC Primer #3 5?-AACAGCCAACATCAACG-3? 571 to 587 Sense Islam et al . (2001b)

Mutagenic Primer Q253H 5?-GTATAAGGCCaTGGACGCTTG-3? 899 to 879 Antisense �/

Mutagenic Primer A284T 5?-TCAGTGCCGGtCGTTAGCCCA-3? 990 to 970 Antisense �/

INCO-DC Primer #4 5?-GCTCGAAGTTGCTCACCC-3? 1247 to 1230 Antisense Islam et al . (2001b)

BD-3VP2-s 5?-CTCAGCCAACATCGATGCCA-3? 820 to 839 Sense �/

BD-3 VP2-as 5?-CGCGCTGGTTGGAATCACAA-3? 1030 to 1011 Antisense �/

a The mutated nucleotide is shown in lower case, the Nco I cleavage site is underlined and the destroyed Nae I cleavage site is in italics.b The nucleotide numbering is based on Bayliss et al . (1990) and modified according to Mundt & Muller (1995).

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cytopathic effects, total RNA was extracted from the infected cells and

subjected to IBDV-specific reverse transcription (RT)-PCR using

INCO-DC Primers #3 and #4 (Table 1). The PCR product was

digested with restriction enzymes Bsp MI, Sac I, Nae I and Nco I to

confirm the identity of the virus. This molecularly cloned tissue culture

adapted vvIBDV was designated as BD-3tc.

Experimental infection

The pathogenicity of BD-3tc was tested in commercial chickens by

experimental infection. The presence of maternally derived antibodies

at the time of inoculation was determined with the enzyme-linked

immunoassay IDEXX FlockChek IBD (IDEXX, Westbrook, Maine,

USA). Titers of 98 to 675 (mean�/290) and 214 to 1215 (mean�/671)

were observed in White Leghorn and Brown Nick chickens used in

Experiment 1 and Experiment 2, respectively. Along with BD-3tc, the

original parent strain BD 3/99 (BD-3wt) (Islam et al ., 2001a) and the

molecularly cloned parental strain (BD-3mc) (Islam et al ., 2001b) were

also used in the study.

Experiment 1. Forty-five unvaccinated 1-day-old White Leghorn chick-

ens obtained from a commercial source were raised in relative isolation.

At 4 weeks of age they were divided into five equal groups housed

separately in cages. Four groups of birds were inoculated with BD-3wt,

BD-3mc, BD-3tc (undiluted) and BD-3tc (diluted 1:100), respectively,

through intraocular and intranasal routes, while the fifth group served

as the uninfected control (Table 2). At days 3, 10 and 14 post-

inoculation (p.i.) three birds from each group were randomly selected,

killed and subjected to routine postmortem examination. The bursa/

body weight ratio was determined at day 10 and day 14 p.i. A part of the

bursa of Fabricius of each sampled bird was fixed in 10% neutral

buffered formalin for histopathological examination.

Experiment 2. A total of 38 1-day-old Brown Nick chickens were

commercially obtained and raised in relative isolation. At 5 weeks of age

they were divided into three groups and placed in three separate houses.

Two groups were inoculated with BD-3wt and BD-3tc, respectively, and

the third group served as uninfected control (Table 2). At days 3, 7 and

14 p.i. three birds from each group were selected randomly, killed and

subjected to routine postmortem examination. The bursa/body weight

ratio was determined on all three occasions. The bursa of Fabricius of

each sampled bird was collected aseptically and fixed in 10% neutral

buffered formalin for histopathological examination as well as RT-PCR.

Histopathology

A part of each sample of the formalin-fixed bursa of Fabricius was

processed for paraffin embedding, sectioned and stained with haema-

toxylin and eosin following standard procedures. On histopathological

examination, a bursal lesion score was determined on the basis of the

following criteria: score 0�/apparently normal lymphoid follicles; score

1�/mild lymphoid depletion indicated by just thinning of the lympho-

cyte population without any sign of focal necrosis or remarkable

oedema; score 2�/moderate lymphoid depletion along with focal

necrosis and interfollicular oedema; score 3�/severe lymphoid deple-

tion virtually leaving no lymphocyte but only reticular cells and

proliferating fibrous tissue; and score 4�/atrophy of follicles usually

with cystic spaces, infolding of epithelium and marked fibroplasia.

RNA extraction from bursal tissue

For RNA isolation, the RNeasy Mini Kit (Qiagen, Hilden, Germany)

was used. Briefly, formalin-fixed bursal samples collected from birds in

Experiment 2 at days 3, 7 and 14 p.i. were rinsed twice with PBS.

Approximately 100 mg samples were ground in a mortar with sterile sea

sand and 4 ml buffer RLT (Qiagen) containing 1% b-mercaptoethanol.

The homogenate was centrifuged at 400�/g for 1 min. A volume of 600

ml was collected from the supernatant, centrifuged at 17,000�/g for 3

min to remove coarse cellular material, and RNA was isolated as

recommended by the supplier of the kit.

RT-PCR combined with restriction enzyme analysis

An RT-PCR protocol combined with restriction enzyme analysis was

developed to detect viral RNA from formalin-fixed bursal material and

to differentiate between BD-3wt (wt-phenotype) and BD-3tc (tc-

phenotype). Primers BD-3VP2-s and BD-3VP2-as (Table 1) amplify a

211 bp RT-PCR product (nucleotide positions 690 to 900 in the VP2

gene according to Bayliss et al. [1990]) enclosing the two point

mutations for the amino acid substitution at positions 253 and 284.

For RT-PCR, the Qiagen OneStep RT-PCR-Kit (Qiagen) was used as

recommended by the supplier with minor modifications. A premix was

prepared as follows: 10 ml of 5�/Qiagen OneStep RT-PCR Buffer, 10 ml

Q-Solution, 2 ml dNTPs (10 mM each), 100 pmol each primer, 10 u

RNasin† (Promega, Mannheim, Germany), and 2 ml Qiagen OneStep

RT-PCR Enzyme Mix were mixed in a total volume of 40 ml. Volumes

of 10 ml total RNA were denatured by heating at 958C for 5 min

followed by snap freezing in liquid nitrogen. Then the premix was added

and the RNA was reverse transcribed by heating at 508C for 30 min.

PCR started with an initial denaturation step of 15 min at 958C. The

temperature profile of each of the following 40 cycles consisted of 30 sec

at 948C for denaturation, 30 sec at 588C for annealing and 60 sec at

728C for elongation. The reaction was terminated by a final elongation

phase of 10 min at 728C. Volumes of 5 ml amplification products were

separated on a 2% agarose gel stained with ethidium bromide.

A second series of 5 ml aliquots of the RT-PCR products was used for

restriction enzyme analysis by digestion with Nae I (Promega, Man-

nheim, Germany) and Nco I (New England Biolabs, Frankfurt am

Main, Germany), respectively, according to the recommendations of the

suppliers. The cleavage products were separated on a 2% agarose gel

stained with ethidium bromide.

Cloning of RT-PCR products and sequencing

RT-PCR products obtained from Bd-3tc-infected bursae harvested 3

days p.i. were cloned using the TOPO TA Cloning† protocol (Invitro-

gen, Karlsruhe, Germany) as recommended by the supplier. Fifty white

or light blue colonies were grown overnight in LB medium. DNA from

each of these colonies was extracted using the Qiagen Plasmed Mini Kit

(Qiagen) and digested with Nae I and Nco I as described previously.

The nucleotide sequences of purified plasmids (Qiagen plasmid mini

kit) were determined using M13 Reverse Primer and an automated 377

ABI DNA Sequencer and together with the Dye Terminator Cycle

Sequencing Kit (Applied Biosystems Inc., Foster City, CA, USA).

Table 2. Design for experimental infection

Experiment Group Inoculum Number of birds Dose

1 I BD-3wt 9 100 ml of 20% bursal homogenate (103.5 EID50)

II BD-3mc 9 100 ml of 20% bursal homogenate (103.1 EID50)

III BD-3tc (undiluted) 9 100 ml undiluted tissue culture fluid (104.0 TCID50)

IV BD-3tc (diluted 1:100) 9 100 ml diluted (1:100) tissue culture fluid (102.0 TCID50)

V Uninfected control 9 �/

2 I BD-3wt 20 100 ml of 20% bursal homogenate (104.0 EID50)

II BD-3tc 9 100 ml tissue culture fluid (103.0 PFU)

III Uninfected control 9 �/

EID50, median embryo infective dose; PFU, plaque forming units.

Reversion of attenuated vvIBDV 183

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Nucleotide sequences of the 211 bp inserts (nucleotide positions 820 to

1030, numbering based on Bayliss et al . [1990] and modified according

to Mundt & Muller [1995]), well readable in all cases and submitted to

the Genbank database (accession numbers AY362978 and AY362979),

were aligned using the Clustal W method of the MegAlign program

(DNA Star Inc., Wisconsin, WI, USA).

Results

Rescue and molecular characterization of BD-3tc

The CEF cells co-transfected with capped RNAtranscripts of cloned cDNA of both segments ofBD-3/99 having two mutations in the VP2 gene tointroduce amino acid substitutions at positions 253(Q0/H) and 284 (A0/T) showed IBDV-specificimmunofluorescence at 24 h post-transfection (datanot shown). Extensive cytonecrotic changes wereobserved from 48 h post-transfection, which prob-ably represented the effects of transfection as wellas virus-induced cytopathic effect. However, whenthe supernatant of the transfected cells was pas-saged in fresh CEF cells, a clear cytopathic effectwas noticed by 48 h p.i. RNA isolated from theCEF cells infected with the rescued virus (BD-3tc)tested positive in IBDV-specific RT-PCR. Cleavageof the PCR product by BspMI but not by SacI(Figure 1, lanes 1 to 4) indicated that the rescuedvirus was vvIBDV like (Zierenberg et al ., 2001).Furthermore, disappearance of the NaeI site andintroduction of an NcoI site confirmed that theintroduced mutations were retained by BD-3tc(Figure 1, lanes 5 to 8).

Clinical manifestations, morbidity and mortality

Experiment 1. All birds inoculated with BD-3wtand BD-3mc developed clinical disease by day 3and day 4 p.i., respectively, which was characterizedby marked depression, ruffled feathers and diar-rhoea. In two BD-3tc inoculated groups, only twoand three birds showed transient depression for 1or 2 days starting at day 8 p.i. There was no

mortality in any group. The birds of the uninocu-lated control group remained healthy throughoutthe experiment.

Experiment 2. In the group of birds inoculated withBD-3wt, 75% (15/20) showed clinical signs ofillness by days 3 and 4 p.i.; the signs were similarto those observed in Experiment 1, but two birdsout of 20 (10%) died on days 5 or 6 p.i. BD-3tc-inoculated birds did not show any clinical signs.The birds of the uninfected control group remainedhealthy throughout the experiment.

Gross pathology

The severity of gross lesions varied between thegroups, and between the birds within a group, at aparticular time point of observation. In Experiment1, following inoculation with BD-3wt and BD-3mc,marked oedematous swelling of the bursa ofFabricius, occasionally with congestion and hae-morrhages, was seen at day 3 p.i., but the bursaappeared highly atrophied at day 10 and day 14 p.i.In BD-3tc groups, the bursa was slightly oedema-tous and swollen at day 3 p.i., almost normal at day10 p.i., but atrophied at day 14 p.i. Petechialhaemorrhages in the breast and thigh muscle wereseen in some birds on all three occasions in the BD-3wt-inoculated and BD-3mc-inoculated groups. InExperiment 2, the gross lesions were similar tothose observed in Experiment 1. However, inExperiment 2 the bursal atrophy seemed to appearfaster and become more pronounced, few birdsshowing slight atrophy even at day 3 p.i.

Bursa/body weight ratio

The bursa/body weight ratios, determined at days10 and day 14 p.i., in Experiment 1 and at day 3,day 7 and day 14 p.i. in Experiment 2, are presentedin Figure 2. In Experiment 1, birds of the BD-3wt-inoculated and BD-3mc-inoculated groups hadsignificantly reduced bursa/body weight ratios atday 10 p.i. However, at day 14 p.i., markedlyreduced bursa/body weight ratios were recordedin all four inoculated groups. The reduction washighest in the BD-3wt group, followed by the BD-3mc, BD-3tc (undiluted) and BD-3tc (diluted)groups. In Experiment 2, bursa/body weight ratiosof the BD-3wt and BD-3tc groups were slightlyreduced at day 3 p.i., declining further at day 7 p.i.,although the reduction was much greater in theBD-3wt group. However, at day 14 p.i., almostsimilar markedly reduced bursa/body weight ratioswere recorded in both BD-3wt and BD-3tc groups.

Histopathology and bursal lesion score

In general, histopathological lesions in the bursa ofFabricius of the inoculated birds were characterizedby lymphoid depletion in the bursal follicles,presence of necrotic cellular masses along with

Figure 1. Agarose gel electrophoresis of the RT-PCR products

of BD-3mc (lanes 1, 3, 5 and 7) and BD-3tc (lanes 2, 4, 6 and 8)

after digestion with the restriction enzymes Bsp MI (lanes 1 and

2), Sac I (lanes 3 and 4), Nae I (lanes 5 and 6) and Nco I (lanes

7 and 8). M, DNA size marker (l-dV1 DNA digested with

Hae III).

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interfollicular oedema and infiltration of hetero-phils and macrophages, cystic atrophy of follicles,infolding of the bursal epithelium and fibroplasia.However, the temporal development and the sever-ity of the lesions varied with the virus strains used.At day 3 p.i., moderate to severe lymphoid deple-tion, interfollicular oedema, and active lymphoidnecrosis were seen in the BD-3wt and BD-3mcgroups. Lesions to a similar extent were not seen inBD-3tc groups until day 10 p.i. (Experiment 1) orday 7 p.i. (Experiment 2), when BD-3wt-inoculatedand BD-3mc-inoclated groups already started todevelop cystic follicular atrophy. Regeneration ofbursal follicles was seen only in BD-3tc-inoculatedgroups at day 14 p.i. in both experiments (Table 3).

Bursal lesion scores of individual birds indifferent groups, calculated on the basis of thepreset criteria at different time-points of observa-tions, are presented in Table 3. At day 3 p.i., BD-3wt-inoculated and BD-3mc-inoculated birds hadremarkable bursal lesion scores. However, at day 10

(Experiment 1) or day 7 (Experiment 2) and day 14p.i., all the inoculated groups had high lesionscores. In general, the lesion score was highest inthe BD-3wt group, followed by the BD-3mc andBD-3tc groups.

Detection and characterization of viral RNA

An RT-PCR product with the expected size of 211bp was obtained with all bursal samples of theinfected chickens (data not shown). The NaeI andNcoI restriction enzyme analysis of the RT-PCRproducts obtained from chickens in Experiment 2 isshown in Figure 3. As expected, at day 3 p.i., RT-PCR products obtained from chickens inoculatedwith BD-3tc were not cleaved by NaeI but by NcoI(lanes 1 and 2, tc-phenotype), whereas those fromBD-3wt were cleaved by NaeI but not by NcoI(lanes 3 and 4, wt-phenotype). Nearly the sameresults were observed at day 7 p.i. However, in thecase of RT-PCR products obtained from chickensinoculated with BD-3tc, two faint bands weredetected after digestion with NaeI (lane 5) and anuncleaved product after digestion with NcoI (lane6) indicating the simultaneous presence of the tc-phenotype and the wt-phenotype. Remarkably, atday 14 p.i. RT-PCR products of the wt-phenotypeonly were detected in both groups (lanes 9 to 12).

Proof of reversion

These results were considered as reversion of BD-3tc to the wt-phenotype in the bursa of infectedchickens. To demonstrate this reversion, RT-PCRproducts obtained from BD-3tc-infected bursaecollected at day 3 p.i. were cloned, and 50 cloneswere investigated by restriction enzyme analysiswith NaeI and NcoI as exemplified for clones 40 to42 in Figure 4. Of these, 49 clones showed thepattern of the tc-phenotype (NaeI-negative, lanes 1and 5; NcoI-positive, lanes 2 and 6) However, theanalysis of one clone (clone 41) exhibited anexceptional pattern (NaeI-positive, lane 3; NcoI-positive, lane 4), indicating that the amino acid atposition 284 in VP2 had reverted to the wild-type

Figure 2. Average bursa/body weight ratios in different groups

determined at days 10 and 14 p.i. in Experiment 1 (top) and at

days 3, 7 and 14 p.i. in Experiment 2 (bottom).

Table 3. Bursal lesion score of individual birds at days 3, 7 or 10 and day 14 p.i.

Group 3 days p.i. 10 days p.i. 14 days p.i.

Experiment 1 BD-3wt 2, 3, 3 4, 4, 4 4, 4, 4

BD-3mc 1, 2, 2 3, 4, 4 4, 4, 4

BD-3tc (undiluted) 0, 1, 1 3, 3, 4 3, 4a, 4a

BD-3tc (diluted) 0, 1, 1 3, 3, 4 3, 3a, 4a

Control 0, 0, 0 0, 0, 0 0, 0, 0

Experiment 2 3 days p.i. 7 days p.i. 14 days p.i.

BD-3wt 3, 3, 3 3, 4, 4 3, 4, 4

BD-3tc 1, 1, 2 3, 3, 4 3, 4a, 4a

Control 0, 0, 0 0, 0, 0 0, 0, 0

The criteria of scoring lesions are described in detail in Materials and methods.a Regeneration of the bursal follicle was observed.

Reversion of attenuated vvIBDV 185

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phenotype (T284A). This was confirmed by se-quencing of inserts in clones 40 and 41, as clone 41had G instead of A at nucleotide position 980(Figure 5). None of the clones showed a patterncorresponding to the wt-phenotype (NaeI-positive,

NcoI-negative), indicating the absence of anycontamination.

Discussion

BD-3wt was originally isolated from an outbreakof acute IBD in a poultry farm in Mymensingh,Bangladesh in 1999 (BD-3/99) and the isolate wasfound to be molecularly and antigenically similarto other vvIBDV from Europe, Asia and Africa(Islam et al., 2001a). The original wild-type strain(BD-3wt) and its molecularly cloned derivative(BD-3mc) (Islam et al ., 2001b) do not grow intissue culture. However, BD-3tc, which was gener-ated after site-directed mutagenesis of two aminoacids (Q253H and A284T) in the capsid proteinVP2, replicates in CEF cell culture. RT-PCRcoupled with restriction enzyme analysis confirmedthat the rescued virus was indeed derived fromvvIBDV and the desired mutations were present inthe genome of BD-3tc.

Recently, van Loon et al . (2002) reported tissueculture adaptation of another vvIBDV strain(UK661) after amino acid exchanges at positions253 and 284 of the VP2, and showed that thismutant (UK661tc) was attenuated for SPF chick-ens. The pathogenicity experiments described herewere performed to know whether the mutations inBD-3tc also caused attenuation and this wasdesigned as a pilot study for planning a detailedexperiment on the evaluation of BD-3tc as apossible vaccine candidate. This study was con-ducted in commercial chickens as the envisagedfuture studies would also be conducted in commer-cial chickens having maternally derived antibodies.

In the first experiment, all birds of the BD-3wt-inoculated and BD-3mc-inoculated groups devel-oped clinical signs typical of IBD (Lukert & Saif,1997) within 2 to 3 days after inoculation. Thechickens inoculated with two dilutions (104 mediantissue culture infectious dose [TCID50] and 102

TCID50) of BD-3tc did not develop the clinicaldisease until day 8 p.i., when two birds showedtransient depression lasting for 1 or 2 days. Thereason for this late transient illness of few birds inthe BD-3tc groups could not be readily explained,so the experimental infection was repeated. In therepeated experiment, 75% of BD-3wt-inoculatedbirds developed clinical disease but BD-3tc-inocu-lated birds remained apparently healthy. BD-3wthad been previously identified as vvIBDV on thebasis of molecular and antigenic analyses (Islam etal., 2001a). Despite the development of typicalclinical disease in the BD-3wt and BD-3mc groups,there was no mortality in the first experiment andonly 10% mortality in the second experiment. Thiswas probably due to the presence of maternallyderived antibodies at the time of inoculation.

Infection with BD-3wt and BD-3mc resulted inmarked atrophy of the bursa of Fabricius at days 7

Figure 3. Agarose gel electrophoresis of the RT-PCR products

of samples collected from the BD-3tc inoculated group (tc) and

the BD-3wt inoculated group (wt) of Experiment 2 at days 3, 7

and 14 p.i. The products were digested with either NaeI (lanes 1,

3, 5, 7, 9 and 11) or Nco I (lanes 2, 4, 6, 8, 10 and 12). M, DNA

size marker (l-dV1 DNA digested with Hae III).

Figure 4. Agarose gel electrophoresis of plasmid DNA obtained

from clones 40, 41 and 42 after digestion with Nae I (lanes 1, 3

and 5) or Nco I (lanes 2, 4 and 6). Fragment sizes obtained with

clones 40 and 42 were: Nae I-negative, 2867, 1275 bp; Nco I-

positive, 2486, 1656 bp (lanes 1, 2, 5 and 6). Fragment sizes

obtained with clone 41 were: Nae I-positive, 2320, 1275, 547 bp;

Nco I-positive, 2486, 1656 bp (lanes 3 and 4). Partially cleaved

high molecular weight bands are also visible. M, DNA size

marker ldV1/PAT.

186 R. Raue et al.

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or 10 and day 14 p.i. Although BD-3tc-infectedbirds remained clinically normal, with the excep-tion of two birds showing late transient clinicalsigns in Experiment 1, they did develop bursalatrophy. However, the course of development ofbursal atrophy was slower in the case of BD-3tc.Histopathological examination of the bursa ofFabricius and scoring of lesions revealed variationsin the course of development and the severity ofbursal lesions in different groups. The lesionsdeveloped faster and were more severe in theBD-3wt and BD-3mc groups as compared withthe BD-3tc groups. Interestingly, the very acutetype of lesions, such as focal necrosis of lympho-cytes and interfocllicular oedema, usually found 3to 4 days after inoculation, was observed at days 7and 10 and even day 14 p.i. in some of the BD-3tc-infected birds.

The results of the present study would indicatethat tissue culture-adapted strain BD-3tc waspartially attenuated for commercial chickens, whichwas characterized by slower and/or subclinicalcourse of the disease, milder pathological lesionsand quick regeneration of the bursal follicles (Table3). However, delayed onset of clinical signs in somebirds and delayed development of acute type bursallesions in some of the BD-3tc-infected birds alsoraised the question of reversion in BD-3tc duringreplication in the birds.

Due to the high error rates of RNA-dependentRNA polymerases (e.g. with error rates of 0.2, 0.8and 1.1 per replication round and per genome inthe case of polio-1 virus, foot and mouth diseasevirus and vesicular stomatitis virus, respectively;Eigen, 2000), reversion is well known in RNA

viruses. Reversion has been defined as mutationthat results in a change from a mutant genotype tothe original wild-type genotype (Condit, 2001).Reversion of an attenuated phenotype to a patho-genic wild-type has been reported for a number ofanimal viruses (e.g. poliovirus) (Macadam et al. ,1989), porcine reproductive and respiratory syn-drome virus (Nielsen et al. , 2001) and Venezuelanequine encephalitis virus (Aronson et al. , 2000).Back mutations have also been described in double-stranded RNA viruses (Coombs et al. , 1994;Ramig, 1998). With the results of the investigationsreported here, reversion in the case of IBDV isdemonstrated for the first time. The phenotypescaused by mutation can revert in one of two ways:by reversion of the original mutation to the wild-type sequence or by mutation at a second site.Second-site mutation might be excluded by theresults of sequence analysis of RT-PCR products,which revealed reversion at nucleotide position 980(A0/G) where G had been substituted by A before.

These results demonstrate that the change fromthe tc-phenotype to the wt-phenotype in BD-3tc-infected birds was due to reversion during replica-tion in the bursa of Fabricius. In isolated Blymphocytes IBDV replication is initiated as soonas 90 min after infection (Muller & Becht, 1982)and viral progeny is formed after 6 to 8 h(Cursiefen et al. , 1979). These data indicate thatseveral rounds of IBDV replication take place inthe bursa of infected chickens, considerably in-creasing the risk of reversion. Therefore, live IBDVvaccines have to be investigated intensively withregard to reversion and, consequently, safety (Jung-back et al. , 1999).

Figure 5. Alignment of the nucleotide sequences obtained from two cloned RT-PCR products (clone 40 and clone 41) corresponding to

nucleotide positions 820 to 1030 of the VP2 gene (nucleotide numbering based on Bayliss et al. (1990); modified according to Mundt &

Muller [1995]). The alignment was performed using the Clustal W method. The nucleotide exchange is boxed, primer locations are in

Reversion of attenuated vvIBDV 187

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Acknowledgements

M. R. Islam was supported by the Georg ForsterResearch Fellowship Programme of the Alexandervon Humboldt Foundation, Germany; and M.N.Islam was supported by the National Science andTechnology Fellowship Programme of the Ministryof Science and Technology, Bangladesh. The in-vestigations were further supported by the Eur-opean Commission: INCO-DC Concerted ActionContract No. ERBIC18CT980330 (‘Acute Infec-tious Bursal Disease in Poultry: Epidemiologyof the Disease, Basis of Virulence and Improve-ment of Vaccination), and COST Action 839 �/

(‘Immunosuppressive Viral Diseases of Poultry’).

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RESUME

Reversion d’un virus de la bursite infectieuse, tres virulent mais attenue

partiellement par genie moleculaire, chez des poulets de chair durant

l’infection

Un virus de la bursite infectieuse aviaire (BD-3tc) adapte a la culture

cellulaire et clone moleculairement a ete genere a partir d’une souche

tres virulente par une approche de genetique reverse suivant une

mutagenese dirigee (Q253H et A284T de VP2). La pathogenicite du

virus BD-3tc a ete etudiee chez des poulets de chair. La souche sauvage

BD-3wt et la souche parentale clonee moleculairement (BD-3mc) ont

ete etudiees en comparaison. Chez les animaux inocules avec la souche

BD-3tc, l’evolution subclinique de la maladie, avec des lesions

pathologiques moins severes, plus tardives et qui ont ete suivies par

une regeneration folliculaire rapide dans la bourse de Fabricius laisse

supposer que les substitutions de ces acides amines rendent la souche

BD-3tc partiellement attenuee. Cependant, une atrophie severe de la

bourse a ete observee 14 jours apres inoculation. Une RT-PCR couplee

a l’analyse d’une restriction enzymatique a revele que les deux points de

mutation de la souche BD-3tc ont reverse 14 jours apres inoculation.

Des investigations complementaires ont demontre que le codon pour

l’acide amine en position 284 avait deja reverse au phenotype sauvage

(T284A) trois jours apres inoculation.

ZUSAMMENFASSUNG

Reversion eines gentechnisch hergestellten, teilweise attenuierten hochvir-

ulenten Stammes des Virus der infektiosen Bursitis nach der Infektion

nicht-spezifiziert pathogen freier Huhner

Unter Verwendung reverser Genetik wurde aus einem hochvirulenten

Stamm des Virus der infektiosen Bursitis ein Zellkultur-adaptierter

Stamm (BD-3tc) hergestellt, in dem mittels gerichteter Mutagenese die

Aminosauren Q253H und A284T in VP2 ausgetauscht worden sind.

Die Pathogenitat von BD-3tc wurde in nicht-spezifiziert pathogen freien

Huhnern getestet. Der Wildtyp-Stamm (BD-3wt) und der gentechnisch

hergestellte Elternstamm (BD-3mc) wurden in die Untersuchungen

einbezogen. Der subklinische Verlauf der Krankheit mit verzogert

einsetzenden und milderen pathologischen Veranderungen der Bursa

Fabricii, gefolgt von einer schnellen Follikel-Regeneration in den BD-

3tc-infizierten Huhnern, ließen vermuten, dass der Austausch der

genannten Aminosauren zu einer teilweisen Attenuierung des Stammes

BD-3tc gefuhrt hatte. Jedoch wurde 14 Tage nach der Inokulation eine

schwere Atrophie der Bursa beobachtet. Untersuchungen mittels RT-

PCR und nachfolgenden Restriktionsenzymanalysen ergaben, dass

beide Punktmutationen in BD-3tc revertiert waren. Weitere Untersu-

chungen zeigten, dass die Reversion fur das Codon der Aminosaure an

Position 284 zum Phenotyp des Wildtyps (T284A) bereits drei Tage

nach der Inokulation eingetreten war.

RESUMEN

Reversion de un virus de bursitis infecciosa aviar muy virulento,

parcialmente atenuado mediante ingenierıa genetica, durante una infec-

cion en pollos comeciales

Un virus de bursitis infecciosa aviar adaptado a cultivo celular y

molecularmente clonado (BD-3tc) fue generado a partir de una cepa

muy virulenta una aproximacion con genetica reversa seguido de site-

directed mutagenesis (Q253H y A284T en VP2). La patogenicidad de

BD-3tc fue probada en pollos comerciales. Se incluyeron la cepa

original (BD-3wt) y la clonada molecularmente (BD-3mc) para poder

ser comparadas. El curso subclınico de la enfermedad, con lesiones

tardıas y leves seguidas de una rapida regeneracion folicular de la bolsa

de Fabricio en las aves inoculadas con BD-3tc, sugieren que estas

sustituciones aminoacıdicas atenuaron parcialmente a BD-3tc. Aun ası,

se observo una atrofia grave de la bolsa a los 14 dıas tras la inoculacion.

La RT-PCR juntamente con el analisis con enzimas de restriccion revelo

que ambas mutaciones puntuales en BD-3tc revirtieron 14 dıas tras la

inoculacion. En investigaciones posteriores se demostro que el codon

para la posicion aminoacıdica 284 habıa ya revertido al fenotipo

original (T284A) tres dıas despues de la inoculacion.

Reversion of attenuated vvIBDV 189

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