Molecular Determinants of Virulence, Cell Tropism, and Pathogenic Phenotype of Infectious Bursal Disease Virus

JOURNAL OF VIROLOGY,0022-538X/01/$04.00�0 DOI: 10.1128/JVI.75.24.11974–11982.2001

Dec. 2001, p. 11974–11982 Vol. 75, No. 24

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Molecular Determinants of Virulence, Cell Tropism, andPathogenic Phenotype of Infectious Bursal Disease Virus

MEGGIN BRANDT,1,2 KUN YAO,2† MEIHONG LIU,2,3 ROBERT A. HECKERT,3

AND VIKRAM N. VAKHARIA1,2,3*

Molecular and Cell Biology Program,1 Center for Agricultural Biotechnology of University of Maryland BiotechnologyInstitute,2 and Virginia-Maryland Regional College of Veterinary Medicine,3 University of Maryland,

College Park, Maryland 20742

Received 4 April 2001/Accepted 20 August 2001

Infectious bursal disease viruses (IBDVs), belonging to the family Birnaviridae, exhibit a wide range ofimmunosuppressive potential, pathogenicity, and virulence for chickens. The genomic segment A encodes allthe structural (VP2, VP4, and VP3) and nonstructural proteins, whereas segment B encodes the viral RNA-dependent RNA polymerase (VP1). To identify the molecular determinants for the virulence, pathogenicphenotype, and cell tropism of IBDV, we prepared full-length cDNA clones of a virulent strain, IrwinMoulthrop (IM), and constructed several chimeric cDNA clones of segments A and B between the attenuatedvaccine strain (D78) and the virulent IM or GLS variant strain. Using the cRNA-based reverse-genetics systemdeveloped for IBDV, we generated five chimeric viruses after transfection by electroporation procedures in Veroor chicken embryo fibroblast (CEF) cells, one of which was recovered after propagation in embryonated eggs.To evaluate the characteristics of the recovered viruses in vivo, we inoculated 3-week-old chickens with D78,IM, GLS, or chimeric viruses and analyzed their bursae for pathological lesions 3 days postinfection. Virusesin which VP4, VP4-VP3, and VP1 coding sequences of the virulent strain IM were substituted for thecorresponding region in the vaccine strain failed to induce hemorrhagic lesions in the bursa. In contrast,viruses in which the VP2 coding region of the vaccine strain was replaced with the variant GLS or virulent IMstrain caused rapid bursal atrophy or hemorrhagic lesions in the bursa, as seen with the variant or classicalvirulent strain, respectively. These results show that the virulence and pathogenic-phenotype markers of IBDVreside in VP2. Moreover, one of the chimeric viruses containing VP2 sequences of the virulent strain could notbe recovered in Vero or CEF cells but was recovered in embryonated eggs, suggesting that VP2 contains thedeterminants for cell tropism. Similarly, one of the chimeric viruses containing the VP1 segment of the virulentstrain could not be recovered in Vero cells but was recovered in CEF cells, suggesting that VP1 contains thedeterminants for cell-specific replication in Vero cells. By comparing the deduced amino acid sequences of theD78 and IM strains and their reactivities with monoclonal antibody 21, which binds specifically to virulentIBDV, the putative amino acids involved in virulence and cell tropism were identified. Our results indicate thatresidues Gln at position 253 (Gln253), Asp279, and Ala284 of VP2 are involved in the virulence, cell tropism,and pathogenic phenotype of virulent IBDV.

Infectious bursal disease virus (IBDV) belongs to the genusAvibirnavirus and is a member of the family Birnaviridae (1, 9,10). The genome of IBDV consists of two segments of double-stranded RNA, which are packaged in a nonenveloped icosa-hedral shell 60 nm in diameter. The larger segment, A, is 3,261nucleotides long, and it encodes a 110-kDa precursor proteinin a single large open reading frame (ORF), which is cleavedby autoproteolysis to yield mature VP2, VP3, and VP4 proteins(1, 12). VP2 and VP3 are the major structural proteins of thevirion, whereas VP4 is a minor protein involved in the process-ing of the precursor protein (3, 12, 13, 28). VP2 is the majorhost-protective immunogen of IBDV and contains the deter-minants responsible for causing antigenic variation (6, 11, 40).VP3 is a group-specific antigen and forms a complex with VP1,which may have an essential role for the morphogenesis of

IBDV particles (5, 19). Segment A also encodes a 17-kDanonstructural (NS) protein from a small ORF which precedesand partly overlaps the large ORF (35). This NS protein isdetected only in IBDV-infected cells, and it is not required forviral replication but plays an important role in pathogenesis(24, 43). The smaller segment, B, is 2,827 nucleotides long, andit encodes VP1, a 97-kDa protein having RNA-dependentRNA polymerase activity (36). This protein is covalently linkedto the 5� ends of the genomic RNA segments (34).

IBDV infects the precursors of antibody-producing B cells inthe bursa of Fabricius (BF), which can cause severe immuno-suppression and mortality in young chickens (2, 15). Viruses ofserotype I are pathogenic to chickens, whereas serotype IIviruses are avirulent for chickens (21). IBDV isolates of sero-type I display a wide range of immunosuppressive potential,pathogenicity, and virulence for chickens. Classic IBDV strainsisolated from the United States in the early 1960s, such as theEdgar, 2512, and Irwin Moulthrop (IM) strains, induce hem-orrhagic lesions accompanied by near-total B-cell follicle de-pletion and cause between 30 and 60% mortality in light-breedchickens. In the late 1980s, Delaware and GLS variant viruses,which cause rapid atrophy of the bursa without the accompa-

* Corresponding author. Mailing address: Center for AgriculturalBiotechnology, 6126 Plant Sciences Building, University of Maryland,College Park, MD 20742. Phone: (301) 405-4777. Fax: (301) 314-9075.E-mail: [email protected].

† Present address: Wyeth-Lederle Vaccines and Pediatrics, Mari-etta, PA 17547.

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nying inflammation, hemorrhage, or mortality caused by theearlier classical strains, were isolated from the Delmarva Pen-insula (32, 33). In the mid 1990s, “very virulent” strains ofIBDV which cause �70% mortality in chickens emerged inseveral European and Asian countries (6, 18, 37). To distin-guish the very virulent strains from the classic vaccine strains,a monoclonal antibody (MAb) was generated against the vir-ulent IM strain (22). This MAb 21 recognizes all the veryvirulent IBDV strains tested to date, but it does not react ifthese viruses are adapted in tissue culture (22, 41).

Earlier studies have shown that very virulent strains ofIBDV lose their virulence potential after serial passage innon-B lymphoid chicken cells (42). Comparison of the deducedamino acid sequences of the very virulent (OKYM) and atten-uated (OKYMT) strains showed specific amino acid substitu-tions within the hypervariable region of the VP2 protein. How-ever, due to the lack of a reverse-genetics system that cangenerate virulent IBDV, it was difficult to pinpoint the aminoacids involved in virulence and cell tropism. By carrying outsite-directed mutagenesis of residues 279 and 284 in VP2, Limand coworkers demonstrated that very virulent IBDV could beadapted to chicken embryo fibroblast (CEF) cell culture (17).Similarly, Mundt reported that residues 253 and 284 of theVP2 protein of the variant virus are necessary for tissue cultureinfectivity (23). However, none of these viruses were tested inchickens to verify the role of these residues in IBDV virulenceand pathogenicity. In a recent study, Boot and coworkers res-cued a very virulent IBDV, using a fowlpox-based reverse-genetics system, and demonstrated that VP2 is not the soledeterminant of the very virulent phenotype (4). However, ex-cept for VP2, the possible role of viral proteins in virulence,cell tropism, and the pathogenic phenotype has not yet beendetermined.

Therefore, in order to identify the viral protein(s) of IBDVthat is involved in virulence, cell tropism, and the pathogenicphenotype, we constructed chimeric clones between the atten-uated vaccine strain D78 and either the virulent (IM) or thevariant (GLS) strain by exchanging VP2-, VP4-, VP4 and -3, orVP1-encoding cDNA fragments. Using the cRNA-based re-verse-genetics system for IBDV, we recovered five chimericviruses, including the virulent one, which contains the epitoperecognized by MAb 21 (virulence marker). In this report, wedescribe the characteristics of these recovered viruses in vitroand in vivo and identify the protein(s) and putative amino acidresidues involved in virulence, cell tropism, and the pathogenicphenotype.

MATERIALS AND METHODS

Cells, viruses, and hybridomas. Vero cells were maintained in M199 mediumsupplemented with 5% fetal bovine serum (FBS) at 37°C in a humidified 5% CO2

incubator and were used for propagation of the virus and transfection experi-ments. Primary CEF cells were prepared from 10-day-old embryonated eggs(SPAFAS, Inc., Storrs, Conn.) as described previously (25). Secondary CEF cellswere maintained in a growth medium consisting of M199-F10 (50%-50% [vol/vol]) and 5% FBS and were used for transfection, virus titration, immunofluo-rescence, and plaque assays. Virus stocks were established by serial passage ofthe recombinant viruses in the cell cultures, except one (recombinant IMVP2[rIMVP2]), which was propagated in embryonated eggs. The D78 vaccine strainand the recovered chimeric viruses were titrated in secondary CEF cells asdescribed previously (25), whereas rIMVP2 was titrated in eggs. The virulent IMand GLS strains of IBDV were obtained from bursae of infected specific-patho-gen-free chickens and purified as described previously (25). A panel of MAbs,

prepared against various strains of IBDV, was used to characterize IBDV anti-gens by antigen capture–enzyme-linked immunosorbent assay (AC-ELISA), asdescribed previously (33, 39). All classical strains of IBDV reacted with MAbsB69, R63, and B29, whereas MAb 57 recognized only the GLS variant strain. Inaddition, MAb 21 prepared against the IM strain was used, which reacted onlywith the highly virulent IBDV strains (22).

Construction of full-length cDNA clones. All manipulations of DNAs wereperformed according to standard protocols (27). Construction of a full-lengthcDNA clone of IBDV genome segment A of strain D78 (with sequence tags),pUC19FLAD78mut, has been described previously (25). It encodes all of thestructural proteins (VP2, VP4, and VP3), as well as the NS protein (Fig. 1). Thecloning of segment A of IBDV strain IM was achieved by generating fouroverlapping cDNA clones. First, the VP2 region of IBDV segment A was clonedby reverse transcription (RT)-PCR, using the oligonucleotide primers IBDVP2R(5�-CCAATTGCATGGGCTAGG-3�; binding to nucleotide positions 1534 to1551) and BamBV (5�-ACGATCGCAGCGATGACAAACCTG-3�; binding tonucleotide positions 119 to 141). This fragment was cloned into a pCR2.1 vector(Invitrogen) to produce pCRIMVP2. The 5� end of IBDV segment A was clonedby RT-PCR, using the oligonucleotide primers 5�IR (5�-CACAGTCAAAATGTAGGTCGA-3�; binding to nucleotide positions 256 to 277) and A5�D78 (25).Similarly, the 3� end of IBDV segment A was cloned by RT-PCR, using theoligonucleotide primers A3�D78 (25) and 3�IF (5�-CAGATGAAAGATCTGCTTG-3�; binding to nucleotide positions 3011 to 3031). Both of these fragmentswere cloned into pCR2.1 to obtain the plasmids pCRIM5� and pCRIM3�, whichwere used for sequence analysis. The remaining portion of IM segment A, whichencodes VP4 and VP3, was cloned by RT-PCR, using primers IBDVP2F (5�-GCTTCAAAGACATAATCCGG-3�; binding to nucleotide positions 1458 to1477) and BglR (5�-CAAGAGCAGATCTTTCATCTG-3�; binding to nucleo-tide positions 3011 to 3031). The resulting fragment was then cloned in pCR2.1to obtain pCRIMVP43. Plasmids pUC19IMVP2 and pUC19GLSVP2 were pre-pared by replacing an MfeI fragment in plasmid pUC19FLAD78mut with therespective MfeI fragments derived from plasmids pCRIMVP2 and pGLS-5, as

FIG. 1. Schematic presentation of various cDNA constructs of IM,D78, and GLS strains in order to generate plus-sense RNA transcriptsusing T7 RNA polymerase. A map of the IBDV genome segments Aand B, with its coding capacity, is shown at the top (drawn to scale).The open boxes depict the coding regions of the D78 strain, whereasthe solid and shaded boxes represent the coding regions of the IM andGLS strains, respectively. Selected restriction sites, important for theconstruction of chimeric cDNA clones of segment A, are shown. B,BglII; M, MfeI, N, NdeI; S, SpeI; X, XhoI. The use of restrictionenzymes to generate these chimeric clones did not alter the readingframe of the polyprotein and hence did not cause extensions or dele-tions of the VP2 (residues 1 to 512), VP4 (residues 513 to 754), or VP3(residues 755 to 1012) proteins. All of the constructs contain a T7polymerase promoter sequence at the 5� end.

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shown in Fig. 1. The cloning of genomic segment A of IBDV strain GLS and theconstruction of plasmid pGLS-5 have been described previously (39).

A modified pUC19 vector was constructed by digesting the vector with bothNdeI and NarI and then religating it to form pUC19�. Next, a full-length D78clone was constructed using this modified pUC19� vector. The 3� end of thefull-length D78 clone was modified by PCR to produce a plasmid, pUC19�D78F,that contained a KpnI site at the end instead of an EcoRI site. This plasmid wasused as a backbone to construct the additional clones described below.

A full-length clone of IM segment A was constructed in several steps. First,plasmid pUC19IMVP2 was digested with SpeI and BglII to release a 1,530-bpfragment that was further digested with MfeI. This SpeI-MfeI fragment was thencombined with the MfeI-BglII fragment of pCRIMVP43 and cloned back into thepUC19IMVP2 vector to obtain pUC19IMVP243. An RsrII-BglII fragment fromthis plasmid was obtained by digestion with the RsrII and BglII enzymes. Thisfragment was then combined with the BglII-BsrGI fragment of plasmidpCRIM3�NC and ligated into the RsrII-BsrGI-digested pUC19�D78F vector.This created a full-length clone, pUC19�IMA, which contains all of the codingsequence of IM segment A but lacks the two nucleotide changes in the 5�noncoding region of wild-type IM. This construct is not shown in Fig. 1. Toevaluate the role of VP4 in virulence, plasmid pUC19�D78F was digested withthe XhoI enzyme to release the VP4 coding fragment, which was then replacedwith the XhoI fragment, derived from pCRIMVP43 (without altering the readingframe), to create plasmid pUC19�IMVP4. In this chimera, residues at positions517, 588, and 702 of D78 were replaced with residues of the IM strain (Table 1).Similarly, to prove that the determinants of virulence, cell tropism, and patho-genicity do not reside in VP4 and VP3, we constructed clone pUC19�IMVP43by digesting plasmid pUC19�IMA with MfeI and replacing this fragment withthe MfeI portion of pUC19�D78F (Fig. 1). As a result of this chimera, twoadditional residues at positions 923 and 981 of the D78 VP3 protein werereplaced with residues of the IM strain (Table 1).

To construct a cDNA clone of segment B of the virulent IBDV strain IM, twoprimer pairs (B5�-D78 and B5-IPD78, and B3�-D78 and B3-IPD78) were syn-thesized and used for RT-PCR amplification. The sequences of the primers wereidentical to the one used for the construction of the segment B cDNA clone ofstrain D78 (25, 43). Using genomic double-stranded RNA as a template, cDNAfragments were synthesized and amplified by using a kit according to the sup-plier’s protocol (Perkin-Elmer). The amplified fragments were cloned betweenthe EcoRI site of a pCR2.1 vector to obtain plasmids pCRIMB5� and pCRIMB3�.To construct a full-length clone of segment B, the 5�-end fragment of IBDV(from plasmid pCRIMB5�) was first subcloned between the EcoRI and PstI sitesof the pUC19 vector to obtain pUC19IMB5�. Then the 3�-end fragment of IBDV(from plasmid pCRIMB3�) was inserted between the BglII and PstI sites ofplasmid pUC19IMB5� to obtain a full-length plasmid, pUC19IMB, which en-codes the VP1 protein (Fig. 1).

DNA from the above-mentioned plasmids was sequenced by the dideoxy chaintermination method (30), using an automated DNA sequencer (Applied Biosys-tem), and the sequence data were analyzed using PC/Gene (Intelligenetics)software. The integrity of the full-length constructs was tested by an in vitrotranscription and translation coupled reticulocyte lysate system using T7 RNApolymerase (Promega Corp.). The resulting labeled products were separated ona sodium dodecyl sulfate–12.5% polyacrylamide gel and visualized by autora-diography.

Transcription and transfection of synthetic RNAs. To generate synthetic tran-scripts of segments A and B, plasmids of segments A and B were linearized withthe BsrGI and PstI enzymes, respectively, and treated as described previously(25). The linearized DNA was used to produce in vitro transcripts with the T7mMessage mMachine kit (Ambion) according to the manufacturer’s instructions.Briefly, approximately 3 �g of linearized DNA template was added to the

transcription reaction mixture (20 �l) containing 40 mM Tris-HCl (pH 7.9); 10mM NaCl; 6 mM MgCl2; 2 mM spermidine; 0.5 mM (each) ATP, CTP, and UTP;0.1 mM GTP; 0.25 mM cap analog [m7G(5�)ppp(5�)G]; 120 U of RNasin; and150 U of T7 RNA polymerase and incubated at 37°C for 1 h. Equimolar amountsof RNA transcripts of segments A and B (�8 �g each) were directly used totransfect cells by the electroporation technique.

Electroporation of Vero and CEF cells was carried out in accordance with theprotocol supplied by the manufacturer (Bio-Rad, Hercules, Calif.). Approxi-mately 2 � 106 cells and 9 �l of RNA transcripts from each segment were placedin a cuvette with a 0.4-cm gap and incubated on ice for 10 min prior to electro-poration. Electroporation was done using a Genepulser set at 200 V with acapacitance of 960 �F. After the shock, the cuvettes were incubated on ice for5 min, and then the cells were placed in medium supplemented with 10% FBSin a six-well plate. The plate was incubated at 37°C in a humidified 5% CO2

incubator. For all of the transfections, except the one performed withpUC19IMVP2, the medium was changed after 16 h. After 4 days, the cells werefreeze-thawed three times, and the supernatant was passed into a T-25 flaskcontaining confluent CEF or Vero cells with 5% FBS-supplemented medium.The monolayer was examined daily for cytopathic effect. Following electropora-tion of CEF cells with transcripts derived from pUC19IMVP2, the medium waschanged after 6 h and replaced with 1 ml of 10% FBS-supplemented medium.After 24 h, the cells were freeze-thawed twice, and the supernatant was inocu-lated into the chorioallantoic membranes (CAMs) of 11-day-old embryos, asdescribed below. After 6 days, the embryos were examined for lesions, and theCAM was collected for further propagation of virus.

Inoculation of CAM. Eleven-day-old embryos were used for the CAM inocu-lation. Briefly, a hole was punched at the air cell as well as in the side of the egg,where the vein structure is well developed. The air was removed from the air cellwith a syringe. The supernatant (100 to 500 �l) was dropped onto the CAM witha syringe through the hole punched in the side of the egg. Both holes were sealed,and the embryo was incubated for 6 days. The embryo was examined daily forsurvival by candling. Death of the embryo is often a sign of virus infection. After6 days, the embryo was examined for other signs of viral infection, such as lesionson the CAM, lesions on the embryo, or stunting of the embryo’s growth.

Characterization of recovered IBDV. To determine the specificity of the re-covered viruses, CEF cells were infected with the transfectant viruses and theinfected cells were analyzed by immunofluorescence assay with rabbit anti-IBDVpolyclonal serum, as described previously (25). To examine viral structural pro-teins expressed by recovered chimeric viruses, the viruses (including the onepropagated in the CAM) were purified by sucrose gradient centrifugation andanalyzed by Western blotting, as described previously (26, 39). To further char-acterize the recovered virus, RT-PCR was performed on the chimeric virus withthe appropriate primer pair, used to produce the original clone. The resultingPCR product was directly sequenced as described above using one of the primersof the primer pair.

Growth curve of chimeric IBDV. To analyze the growth characteristics ofIBDV, confluent secondary CEF cells (in T-25 flasks) were infected with one ofthe recovered virus stocks (generated after five passages in Vero or CEF cells) ata multiplicity of infection of 0.1. Infected cell cultures were harvested at differenttime intervals, and the titer of infectious progeny was determined by plaque assayon CEF cells as described previously (25). For rIMVP2, which does not propa-gate in tissue culture, a 50% egg infectious dose (EID50) was used to determinethe virus titer. Serial dilutions of the virus were made and then inoculated intoCAMs as described previously. For each dilution, the number of embryos deador showing lesions was determined. Then, and EID50 was determined using theReed-Muench formula.

Chicken inoculation. Three-week-old specific-pathogen-free chickens wereobtained (SPAFAS, Inc.) and housed in isolators. Prior to inoculation, the

TABLE 1. Location of amino acid changes in VP2, VP4, VP3, and VP1 proteins of IBDV strains D78 and IM

Protein Amino acid change (position)

D78 VP2 Gly (76) Val (242) His (253) Thr (270) Asn (279) Thr (284) Arg (330)IM VP2 Ser Ile Gln Ala Asp Ala SerD78 VP4 Tyr (517) Glu (588) Arg (702)IM VP4 His Lys LysD78 VP3 Arg (923) Leu (981)IM VP3 Lys ProD78 VP1 Thr (13) Arg (115) Gly (147) Glu (515) Leu (546) Glu (653) (879)IM VP1 Lys Gly Asp Asp Pro Lys Gln

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chickens were bled, and their sera were tested by ELISA to ensure that they werenegative for IBDV-specific antibodies. Eight groups of chickens were inoculatedby the ocular route with either culture medium or wild-type IM, D78, rIMB,rIMVP43, rIMVP4, GLSVP2, or rIMVP2 virus. For viruses that were able toreplicate in cell culture, a dose of 1,000 PFU was administered to each group ofchickens, whereas a dose of 1,000 EID50 was given to chickens in the IM andrIMVP2 groups. After 3 days, any surviving chickens were humanely killed andthe bursae were excised and bisected. One hemisection was used for RT-PCRassay and AC-ELISA, while the other was fixed and sectioned for histopatho-logical examination.

Identification of recovered viruses by RT-PCR and AC-ELISA. In order todetermine whether the nucleotide sequence in the recovered viruses is of chi-meric origin, total nucleic acids from IBDV-infected CEF cells, CAMs, or bursalhomogenates were isolated and analyzed by RT-PCR, as described above. Seg-ment-specific primers were used for RT of genomic RNA. Following RT, thereaction products were amplified by PCR with the desired segment-specificprimer, and the amplified products were either directly sequenced or cloned intothe pCR2.1 vector and then sequenced as described above. Generally, the nu-cleotide sequence in chimeric viruses was determined twice, once after recoveryin vitro and again after in vivo studies of bursal homogenates. The recoveredviruses were further characterized with a panel of IBDV-specific MAbs in anAC-ELISA, as previously described (40).

Histopathological studies. The bursa tissues were fixed by immersion in 10%neutral buffered formalin. The ratio of fixative to bursa exceeded 10:1. Sevendays later, a cross sectional portion of each bursa was processed through paraffin,stained with hematoxylin and eosin, and examined with a light microscope. Theseverity of the lesion was graded on a scale of 1 to 5, based on the extent of thelymphocyte necrosis, follicular depletion, and atrophy.

Nucleotide sequence accession numbers. The complete nucleotide sequencesof IBDV genome segments A and B of the IM strain have been deposited in theGenBank database under accession no. AY029166 and AY029165, respectively.

RESULTS

Sequence analysis of segments A and B of virulent IBDVstrain IM. We determined the complete nucleotide sequencesof IM IBDV genome segments A and B, including the 5�- and3�-terminal sequences. These segments are 3,261 and 2,827 bplong, respectively, which is identical to the sizes of the seg-ments of strain D78. Comparison of the 5�- and 3�-terminalsequences of both IM segments with those of the D78 strainshowed nucleotide substitutions of A3G and T3C at posi-tions 69 and 80 in segment A and G3A and A3G at positions59 and 69 in segment B, respectively. Comparison of the de-duced amino acid sequences of IM segments A and B withthose of the D78 strain showed 98.82 and 99.32% identity atthe amino acid level, respectively. There are a total of 12 aminoacid substitutions in segment A between IM and D78, of whichseven are located in VP2, three in VP4, and two in the VP3region of the polyprotein (Table 1). Furthermore, there are atotal of seven amino acid changes in segment B between IMand D78, including an additional amino acid residue at the Cterminus of VP1 in the IM strain (Table 1). This suggests thatthe classical strain IM is closely related to the D78 vaccinestrain. A phylogenetic analysis, based on the deduced aminoacid sequences of segments A and B of very virulent or virulentstrains of IBDV, revealed two distinct groups. The first groupconsisted of classical virulent strains, namely, IM, STC, Edgar,and 52/70, that were isolated in the early 1960s and 1970s andcaused about 30% mortality. The second group comprised allvery virulent strains, such as UK661 (United Kingdom),OKYM (Japan), and HK46 (Hong Kong), that were isolated inthe late 1980s and early 1990s and caused �70% mortality(data not shown).

Construction of full-length and chimeric cDNA clones. Toidentify the molecular determinants of the virulence, patho-

genic phenotype, and cell tropism of IBDV, we constructedchimeric cDNA clones between the attenuated vaccine strainD78 and the virulent strain IM or the variant GLS strain.Figure 1 shows the construction of these clones, in which VP2,VP4, and VP4-VP3 coding sequences of IM or GLS weresubstituted in the D78 backbone. The chimeric nature of theseclones was confirmed by DNA sequence analysis. In addition,we also constructed a full-length cDNA clone of IM segment Bto determine whether the RNA polymerase would play a rolein virulence or cell tropism. The clones were constructed witha pUC19 vector, and all contained a T7 promoter sequencepreceding the 5� noncoding regions of the full-length cDNAclones. The functionality of all these clones was tested by invitro transcription-coupled translation reactions, which yieldedprotein products that comigrated with the marker IBDV pro-teins after fractionation on a sodium dodecyl sulfate–12.5%polyacrylamide gel and autoradiography (data not shown).

Transfection and recovery of chimeric viruses. To identifythe protein(s) responsible for the cell tropism, virulence, andpathogenic phenotype of IBDV, we transfected Vero cells byan electroporation procedure with combined plus-sense tran-scripts derived from various plasmids, as shown in Table 2.From six transfection experiments, we recovered three chi-meric viruses (rGLSVP2, rIMVP4, and rIMVP3) and the pa-rental D78 virus, rD78, containing the tagged sequences. Noother viruses could be recovered in Vero cells, even after fiveattempts, when the cRNA transcripts comprised the codingregions of the IM VP1 (pUC19IMB) or VP2 (pUC19IMVP2)protein. Therefore, CEF and Vero cells were transfected inparallel with the transcripts derived from the plasmid pairs (i)pUC19IMB and pUC19FLAD78mut, (ii) pUC19IMVP2 andpUC19D78B, and (iii) pUC19D78B and pUC19FLAD78mut.From these transfections (done in duplicate), the rIMB viruswas recovered only in CEF cells, as shown in Table 2, whereasthe parental rD78 virus was recovered in both Vero and CEFcells, as expected. To determine the presence of infectiousvirus in transfected Vero cells, the cells were freeze-thawedtwice, and the supernatants were used to infect CEF cells. Novirus could be recovered even after four passages in CEF cells.This result suggests that VP1 contains the determinants for

TABLE 2. Generation of chimeric IBDVs after transfection withtranscripts derived from cloned cDNA of segments A and B in Vero

cells, CEF cells, and CAM

Plasmids used fortranscription

TransfectionaRecombinant

virusVero CEF CAM

pUC19D78B � � ND rD78pUC19FLAD78mutpUC19D78B � � ND rGLSVP2pUC19GLSVP2pUC19D78B � � ND rIMVP4pUC19�IMVP4pUC19D78B � � ND rIMVP43pUC19�IMVP43pUC19IMB � � ND rIMBpUC19FLAD78mutpUC19D78B � � �b rIMVP2pUC19IMVP2

a �, virus recovered; �, no virus recovered; ND, not done.b Recovery of virus after transfection in CEF cells and passage in CAM.


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cell-specific replication in Vero cells. Furthermore, therIMVP2 virus (containing the IM VP2 protein) could not berecovered in CEF cells (after at least five attempts), suggestingthat VP2 contains the molecular determinants for cell tropism.Since we could not recover the rIMVP2 virus, we examined thetransient expression of IBDV-specific proteins in CEF cells.Twenty-four hours posttransfection, we detected the presenceof viral proteins by immunofluorescence assay, using anti-IBDV polyclonal serum (data not shown). Therefore, to assessthe presence of infectious virus in transfected CEF cells, thecells were freeze-thawed twice, and the supernatants were usedfor CAM inoculation of 11-day-old embryonated eggs. After 6days, the embryos showed signs of viral infection, and therecovered rIMVP2 virus was propagated in the CAMs forfurther characterization. These results demonstrate that therIMVP2 virus is able to replicate in CEF cells after transfectionbut cannot be propagated further in CEF cells. Since parentalIM or rIMVP2 viruses are able to grow only in B lymphoidcells and not in CEF cells, it suggests that the major determi-nants of B-lymphocyte cell receptors reside in VP2.

Characterization of chimeric viruses. To verify whether therecovered viruses were of recombinant origin, the genomicRNAs were isolated from these viruses and analyzed by RT-PCR using primer pairs specific for segment A or B. Sequenceanalysis of the cloned PCR product confirmed the expectednucleotide sequences in the VP2, VP4, or VP3 genes of thevarious chimeric viruses. However, when the VP1 gene of therecovered rIMB virus was sequenced, it showed reversion oftwo amino acid residues to the parental D78 segment B se-quence. The residues that reverted were Gly3Arg andLys3Glu at amino acid positions 115 and 653, respectively(Table 1). This result indicates that VP1 may play a role incell-specific replication, as we were unable to recover this virusin Vero cells but could recover it in CEF cells after substitution

of the above-mentioned residues. After four passages of therIMB virus in CEF cells, the virus could replicate in Vero cells.

To determine the replication kinetics of rD78, rIMB,rIMVP4, rIMVP43, and rGLSVP2, CEF cells were infectedwith each virus, and their titers were determined by plaqueassay. Figure 2 depicts the growth curve of each virus (ex-pressed as log PFU per milliliter) on different days postinfec-tion. Our results indicate that the final titers of the chimericviruses were approximately the same as that of the recoveredrD78 parent virus (107 PFU/ml). Furthermore, the chimericviruses were purified by sucrose gradient, and their proteinswere analyzed by Western blot analysis using anti-IBDV poly-clonal serum. Qualitatively, viral structural proteins (VP2,VP4, and VP3) produced by the chimeric viruses were identicalto the proteins synthesized by the recovered rD78 virus (datanot shown).

In vivo studies and histopathological examination of thebursa. To assess the virulence and pathogenic phenotype ofthe recovered viruses, groups of 3-week-old chickens were in-oculated with 1,000 PFU of rD78, rIMB, rIMVP4, rIMVP43,or rGLSVP2 virus or with 1,000 EID50 of rIMVP2 or IM virusby the ocular route. Three days postinoculation, the bursa ofeach chicken was excised and analyzed for pathological lesions.Table 3 and Fig. 3 summarize the results of gross pathologyand histopathological examination of bursae obtained fromdifferent groups of chickens. Chickens inoculated with therD78, rIMB, rIMVP4, and rIMVP43 viruses showed no grossbursal lesions but had mild-to-moderate (for rIMVP43) micro-scopic lesions (Table 3 and Fig. 3B to E, respectively). Theseresults suggest the amino acid changes in the VP1, VP4, andVP3 proteins (Table 1) do not contribute to the virulence orpathogenic phenotype of IBDV in our system. However, chick-ens inoculated with rGLSVP2, rIMVP2, and the parental IMstrain exhibited bursal atrophy (rGLSVP2) or gross hemor-

FIG. 2. Growth curves of rD78, rIMB, rIMVP4, rIMVP43, and rGLSVP2 IBDVs. Monolayers of CEF cells were infected with the indicatedviruses at a multiplicity of infection of 0.1 and harvested at the indicated time points, and infectious titers were determined by plaque assay.


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rhagic lesions with 20 to 25% mortality in the rIMVP2 and IMgroups (Table 3). Moreover, the sections derived from thesegroups showed lymphocytic necrosis and follicular (B-lympho-cyte) depletions due to extensive accumulation of mononu-clear cells, resulting in the loss of distinction between thecortex and medulla of the infected bursal section (Fig. 3F toH). In contrast, no gross or microscopic lesions were observedin chickens that were inoculated with the control medium (Fig.3A). These results clearly demonstrate that the virulence andpathogenic-phenotype markers of the U.S. virulent and variantIBDV strains reside in VP2.

Detection of viral antigen from the bursa. To detect thepresence of virus in the bursae of infected chickens, bursaefrom each group were pooled and homogenized in M199 me-dium, and the filtered homogenate was analyzed by AC-ELISAusing a panel of strain-specific IBDV MAbs. Table 4 shows thereactivity patterns of various IBDVs recovered from groups ofinfected chickens. It should be noted that MAb 21 recognizesall the virulent strains tested so far, and it is evident that thisepitope was present in chimeric rIMVP2 virus, which is also

FIG. 3. Histopathological appearance of sections (hematoxylin and eosin) of BFs derived from groups of chickens infected with chimeric virusesat day 3 postinfection. (A) Cortical lymphocytes (dark-gray cells adjacent to connective tissue that separates follicles) and medullary lymphocytes(light-gray cells in follicle centers) in portions of follicles from an uninfected chicken are normal. In addition, the interfollicular connective tissuesare normal. (B, C, and D) Follicles and interfollicular connective tissues from chickens infected with rD78, rIMB, and rIMVP4 viruses, respectively,appear normal and cannot be differentiated from their control counterparts. (E) Slight damage to the medullary lymphocytes in the bursa froma chicken infected with rIMVP43 virus. (F) There is lymphocytic necrosis and heterophilic inflammation in follicles of the bursa from a chickeninfected with rGLSVP2 virus. (G and H) Severe lymphocytic necrosis accompanied by hemorrhagic lesions in bursae from chickens infected withrIMVP2 and IM viruses. Notice the loss of distinction between the cortex and the medulla and the bands of interfollicular connective tissue thatare infiltrated by myriad heterophils and macrophages. (Magnifications, �100).

TABLE 3. Gross and microscopic lesions in BFs from chickensinfected with recovered IBDVs

GroupPathology

Grossa Microscopicb

Control � �(3/3)rD78 � �(4/4)rIMB � �(5/5)rIMVP4 � �(5/5)rIMVP43 � ��(3/5)rGLSVP2 ��; atrophy ��(5/5)rIMVP2 ��; hemorrhagic ��(5/5)IM ��; hemorrhagic ��(4/4)

a �, gross bursal lesion; �, no gross lesion. �� to �� was graded on thedegree of visible, gross bursal lesion formation.

b �, no histological lesion; � to �� was graded on the degree of extent ofmultifocal purulent necrotizing bursitis with follicular depletion. The numbers inparentheses represent the number of bursae with lesions/number of bursaeexamined. The result is representative of two independent experiments.

TABLE 4. Detection of virus in BFs of chickens infected withvarious IBDVs by AC-ELISA and RT-PCR

Group

Results

AC-ELISAa

RT-PCRb

B69 R63 57 21 B29

Control � � � � � �rD78 � � � � � �rIMB � � � � � �IM � � � � � �rIMVP2 � � � � � �rIMVP4 � � � � � �rIMVP43 � � � � � �GLS � � � � � �rGLSVP2 � � � � � �

a BFs from each group were pooled, and bursal homogenates were tested forreactivity with a panel of strain-specific MAbs. �, viral antigen detected byAC-ELISA; �, no viral antigen could be detected by AC-ELISA.

b �, viral RNA was detected by RT-PCR; �, no viral RNA was detected byRT-PCR.


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virulent. Neutralizing MAb 57 is specific for the GLS variant,and this epitope was also mapped to the VP2 protein.

To determine the genetic stability of chimeric IBDVs in vivo,chickens were inoculated with these viruses, and their bursaewere collected 3 days postinfection. Total nucleic acid wasextracted from bursal tissue, and the genomic segments A andB were amplified by RT-PCR using a primer pair specific forsegment A or B. Sequence analysis of the cloned PCR productsconfirmed the expected nucleotide substitutions or changes invarious genes of the chimeric viruses. These results clearlydemonstrate that the chimeric viruses replicated in the bursaeof chickens but did not revert to the parental D78 or IMviruses.

Determinants of cell tropism, virulence, and pathogenicphenotype of IBDV. To identify putative amino acids involvedin cell tropism, and possibly binding to the cell receptor, wecompared the amino acid sequences of the VP2 protein (vari-able region) of D78, IM, and other selected IBDV strains (Fig.4A). Comparison of the D78 sequence (tissue culture) with thebursa-derived IM, Edgar, and STC sequences showed onlyfour amino acid differences at positions 253, 279, 284, and 330,which are common to other bursa-derived UK661, HK46, andOKYM sequences, suggesting the involvement of these resi-dues in cell tropism. To further narrow down the amino acidsinvolved in cell tropism, we constructed another clone(pUC19�D78VP2), in which amino acids at positions 279, 284,and 330 were replaced with the D78 sequence in plasmidpUC19IMVP2 (Table 1). However, transfection of CEF cellswith transcripts derived from clones pUC19�D78VP2 andpUCD78B did not yield a viable virus, implying that residue Qat position 253 (Q253) is also involved in cell tropism. Thisresult is in agreement with the results of Mundt, who demon-strated that a Q3H substitution at position 253 is sufficient toallow tissue culture infectivity of the E/Del virus (23). Further-more, comparison of the bursa-derived GLS sequence ofgenomic segment A with the tissue culture-adapted GLS-5sequence (Fig. 4B) reveals only Q3H and A3T substitutionsat positions 253 and 284, respectively, suggesting the impor-tance of residues Q253 and A284, but not residue S330, in celltropism. Taken together, our results indicate that residuesQ253, D279, and A284 of VP2 are involved in the cell tropismof virulent IBDV strains.

To identify the putative amino acids involved in virulenceand the pathogenic phenotype, we used MAb 21. This MAbrecognizes a conformational epitope in VP2 and reacts onlywith bursa-derived virulent IBDV (22, 41). MAb 21 reacts withrIMVP2 and fails to react with the classic attenuated D78 orother chimeric viruses (rIMVP4 or rIMVP43), which stronglysuggests that the determinants for virulence reside in the VP2region (Table 4). Comparison of the D78 sequence with thebursa-derived IM, Edgar, STC, and UK661 sequences (all ofwhich react with MAb 21) revealed only three amino aciddifferences at positions 253, 279, and 284, which are commonto all other virulent IBDV sequences; this suggests the involve-ment of these residues in virulence (Fig. 4A). In fact, these arethe same residues that are involved in cell tropism. Based onthe reactivity with MAb 21, it appears that residues Q253,D279, and A284 are critical in binding to this MAb and im-parting the pathogenic phenotype to virulent IBDV.

DISCUSSION

Lack of a reverse-genetics system that can generate virulentIBDV has hampered studies of virulence, cell tropism, andpathogenesis. To overcome this limitation, we modified ourtransfection procedure and developed an efficient method oftransfecting Vero or CEF cells by electroporation, followed bypassage in the CAM to recover virus that would not propagatein tissue culture (virulent IBDV). Using this technique, wegenerated five chimeric viruses, including the virulent rIMVP2,and demonstrated that the virulence, cell tropism, and patho-genic-phenotype markers of IBDV reside in VP2. The fact thatwe recovered the virulent virus after transfection in CEF cellsand propagation in embryonated eggs implies that CEF cellsdo not possess the receptors for virulent IBDV, which repli-

FIG. 4. Comparison of the deduced amino acid sequences of se-lected IBDV strains in the VP2 variable region. (A) Comparison of thededuced amino acid sequence of the D78 strain with those of highlyvirulent IBDV strains. IM and Edgar, highly virulent strains from theUnited States; STC, standard challenge strain in the United States;UK661, very virulent strain from the United Kingdom; HK46, veryvirulent strain from Hong Kong; OKYM, very virulent strain fromJapan; OKYMT, attenuated tissue culture virus of virulent OKYMstrain. (B) Comparison of the deduced amino acid sequence of theD78 strain with those of the U.S. variant strains GLS-5 (tissue culture),GLS (bursa-derived), E/Del, and A/Del. The dashes indicate aminoacid identity.


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cates only in B lymphoid cells. In addition, we recovered achimeric virus containing VP1 of the virulent IM strain in CEFcells but not in Vero cells, suggesting that VP1 contains thedeterminants for cell-specific replication in Vero cells, which isnot receptor mediated.

In recent years, a number of investigators have shown thatmutations in the viral genome often lead to changes in thevirulence, pathogenesis, and cell tropism of animal viruses (7,8, 14, 16, 20, 29). For example, a single-amino-acid change inthe capsid protein VP1 of coxsackievirus was responsible forthe virulence phenotype (7). In swine vesicular disease virus (apicornavirus), the genetic determinants of pathogenicity andplaque phenotype were mapped to a single amino acid residuein VP1 and the 2A proteinase, respectively (14). Similarly, thedeterminants of virulence and enteric tropism in transmissiblegastroenteritis virus were mapped to the spike protein of thevirus (29). For rotaviruses of groups A and C, a substitution oftwo or three amino acid residues in the nonstructural proteingene NSP4 was implicated in reduced virulence (8). In the caseof Sindbis virus, the genetic determinants responsible for thepathogenic properties were mapped to the E2 glycoproteinand the 5� noncoding region (16). In our case, the 5� non-coding region of the virulent IM strain does not contain thedeterminants for virulence, since the rIMVP2 virus, lackingthe two nucleotide changes in the 5� noncoding region ofwild-type IM, was still virulent and caused hemorrhagic bur-sal lesions.

To the best of our knowledge, this is the first report dem-onstrating that the polymerase (VP1) of virulent IBDV carriesthe determinant for cell-specific viral replication, since wecould not recover the virus (rIMB) in Vero cells. However,once we recovered this virus in CEF cells, it could be propa-gated in Vero cells. Sequence analysis of the VP1 gene of therIMB virus showed reversion of two amino acid residues atpositions 115 and 653 to those of the D78 vaccine strain, whichsuggests that these residues may be necessary for replication inVero cell culture (Table 1). This finding is somewhat analo-gous to the case of lymphocytic choriomeningitis virus, whereit was shown that a single-amino-acid substitution in the poly-merase gene could enhance viral replication in macrophages(20). Furthermore, our results indicate that the rIMB virusdoes not induce hemorrhagic lesions in the bursae of infectedchickens (Table 3 and Fig. 3C), suggesting that VP1 of the IMstrain is not responsible for virulence or the pathogenic phe-notype, in agreement with the results of Boot et al. (4). Thisresult is also in accord with that for infectious pancreatic ne-crosis virus (another birnavirus), where it was shown by gen-erating reassortants between virulent and avirulent strains thatthe virulence of infectious pancreatic necrosis virus is associ-ated with segment A, which encodes the structural proteins,and not segment B, which encodes VP1 (31).

From earlier studies of IBDV, Yamaguchi and coworkersidentified amino acid residues responsible for attenuation ofthe very virulent OKYM strain by comparing the sequence ofthe virus, which was obtained after serial passage in non-Blymphoid chicken cells (42). Comparison of the deducedamino acid sequences of the very virulent (OKYM) and atten-uated (OKYMT) strains showed specific amino acid substitu-tions within the hypervariable region of the VP2 protein.Based on these findings, Lim and coworkers mutated residues

279 and 284 of VP2 by site-directed mutagenesis and demon-strated that the very virulent HK46 strain of IBDV could beadapted to CEF cell culture (17). Similarly, Mundt reportedthat residues 253 and 284 of the VP2 proteins of the variantvirus are necessary for tissue culture infectivity (23). However,none of these viruses were tested in chickens to verify the rolesof these residues in IBDV virulence and pathogenicity. Ourresults indicate that VP2 carries the major determinant of celltropism in IBDV, since we could not recover the rIMVP2 virusin Vero or CEF cells but were able to recover it after passagein the CAM. This was expected, since virulent IBDV does notgrow in tissue culture and can only replicate in bursal cells.These results imply that bursal cells possess virus receptors inaddition to the one that is present in tissue culture cells whichVP2 interacts with. Furthermore, our results clearly demon-strate that VP2 contains the determinants for the virulence andthe pathogenic phenotype of IBDV. However, these results arein contrast to those reported by Boot and coworkers, whostated that VP2 is not the sole determinant of the very virulentphenotype due to the fact that their recombinant virus con-taining VP2 of the very virulent strain did not induce morbidityor mortality (4). In our case, rIMVP2 virus containing the VP2region of the virulent strain caused hemorrhagic bursal lesionsand mortality equivalent to those of the parental IM virus,suggesting that the virulence and pathogenic-phenotype mark-ers of IBDV reside in VP2. This is further supported by theresults for chimeric rGLSVP2 virus (containing VP2 of thevariant strain), which caused bursal atrophy, a characteristicphenotype of a variant virus (Table 3). Comparison of theGLS-5 sequence with the D78 sequence showed five uniqueamino acid differences at positions 222, 249, 254, 270, and 330(Fig. 4B), which are common to other variant GLS, E/Del, andA/Del sequences, suggesting the involvement of these residuesin the variant phenotype. In earlier studies, we mapped theclassic MAb B69 neutralization epitope (which is absent in thevariant viruses) to residues at positions 222, 249, and 254 (38,41). Therefore, it is apparent that residues T/Q222, K249, andS254 are important in inducing a characteristic phenotype ofthe variant IBDVs.

In conclusion, we have demonstrated that VP1 carries thedeterminants for cell-specific replication of IBDV in Vero cellsand VP2 carries the determinants for cell tropism. The aminoacids involved in binding with the B lymphoid cells are Q253,D279, and A284. Furthermore, the virulence factor of IBDVstrain IM resides in VP2 and, based on the reactivity withconformation-dependent MAb 21, residues Q253, D279, andA284 are most likely involved in maintaining this conformationand inducing the pathogenic phenotype (hemorrhagic lesions)of virulent IBDV. However, it is possible that VP4 and VP3carry additional determinants for virulence, as the rIMVP43virus also induced slightly more microscopic lesions than theparental D78 virus. In addition, the cooperative effect due tothe expression of the 17-kDa NS protein in virulence shouldnot be ruled out, as we have shown that the 17-kDa knockoutmutant of strain D78 is nonpathogenic (43). It would be inter-esting to see whether the NS-deficient mutant of the IM orGLS virus would induce similar pathological lesions and causeimmunosuppression.


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ACKNOWLEDGMENTS

We thank Donald L. Nuss for reviewing the manuscript, and wethank Gerard H. Edwards, Yi Liu, Subbiah Elankumaran, and RubyParamadhas for technical assistance.

This work was supported by a grant from the U.S. Department ofAgriculture (NRICGP 97-02492) to V.N.V.

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Molecular Determinants of Virulence, Cell Tropism, and Pathogenic Phenotype of Infectious Bursal Disease Virus