Residues of 862 , 921 of VP 3 are associated with virulence in infectious bursal disease virus strain Harbin-1

Reverse genetics was used to study the effect of particular amino acids of infectious bursal disease virus (IBDV) on virulence. Using site-directed mutagenesis, altering of two amino acids in VP2 (Q253H, A284T) and VP3 (H783Q, V862M, I921V) in the segment A of a Chinese very virulent IBDV field strain Harbin-1, 4 virus mutants including H253/284, H783/862, H862/921, H921/783 were rescued. To evaluate the characteristics of the recovered viruses in vivo, we inoculated 4-week-old chickens with virus mutants and rescued Harbin1 (rHarbin-1), analyzed their bursae for pathological lesions 4 days postinfection. rHarbin-1 and H783/862, H253/284 caused severe bursal lesion, milder lesion for H862/921, mildest for H921/783. However, H253/284 caused the lowest mortality. The results showed that residue at position Q253, A284 of VP2 and V862, I921 of VP3 gene are involved with virulence, but there is difference between VP2 and VP3’s role in virulence. The ability of 862 and 921 to control virulence in VP3 is stronger than 253 and 284.


INTRODUCTION
Infectious bursal disease (IBD) is a highly contagious disease among young chickens and characterized by the destruction of the bursa of Fabricius.IBD was first described by Cosgrove [1], but in China the first case was reported in 1979 [2].Nowadays IBD has spread worldwide and continues to threat the poultry industry.Infectious bursal disease virus (IBDV) is the causative agent of the disease, belonging to Avibirnavirus genus of the Birnaviridae family [3].Europe had experienced the emergence of very virulent infectious bursal disease virus (vvIBDV) which can cause up to 70% flock mortality [4,5].Meanwhile, vvIBDV infections also have been observed in Asia and in South America [6].
The genome of IBDV consists of two segments of double-stranded RNA (dsRNA), approximately 3.4 kb (segment A) and 2.7 kb (segment B) in length [7].Segment A contains two partially overlapping open reading frames (ORFs).The larger ORF encodes a polyprotein (1,012 amino acids, 110 kDa) that is autocatalytically cleaved to yield the viral proteins pVP2 (VPX) (48 kDa), VP4 (29 kDa) and VP3 (33 kDa).During virus maturation, pVP2 is processed into matured VP2 (41 to 38 kDa), probably resulting from site-specific cleavage of pVP2 by a host cell-encoded protease [8].The smaller ORF encodes the nonstructural protein VP5 (145 to 149 amino acids, 17 kDa).Segment B encodes VP1 (970 kDa) having putative RNA-dependent RNA polymerase activity [9,10].This protein is covalently linked to the 5' ends of the genomic RNA segments or present at a free form [11,12]. VP2 and VP3 are the major structural protein of the virion.The VP2 is the major host-protective antigen of IBDV and contains the determinants responsible for causing antigenic variation [13][14][15].Position 279 and 284 amino acids in the VP2 variable region possibly contribute to virulence of IBDV [16].Residues 253 and 284 of the VP2 protein of the variant virus are necessary for tissue culture infectivity [17].The virulence and pathogenic-phenotype markers of IBDV reside in VP2 and residues at position 253 (Gln), 279 (Asp) and 284 (Ala) of VP2 are involved in the virulence and pathogenic phenotype of virulent IBDV [18][19][20].However, recent study demonstrated VP2 is not the sole determinant of the very virulent phenotype [21].C-terminal part of VP3 may play a decisive role in controlling the virulence [22].VP3 could play an important role in receptor-mediated virus-cell attachment, which implied that VP3 has relation with virulence [23].
In order to verify if VP3 have molecular determinant of virulence for Chinese vvIBDV strain Harbin-1, amino acids in VP3 among Harbin-1, D78 (vaccine strain), TY89 (IBDV serotype II) were aligned, the different amino acids among them were listed in Table 1.TY89 could not infect B lymphocytes, having no virulence to B lymphocytes, and D78 has mild virulence to B lymphocytes.Based on the result of alignment the amino acids in VP3 that maybe involved in virulence could be found.Position 783 and 862 in Harbin-1 have different amino acids from D78, however, position 921 is different from TY89.To prove their role in virulence, position 783, 862 and 921 in VP3 were mutated subsequently to obtain the combination of two points mutation.As a control, position 253 and 284 in VP2 hypervarible region was mutated at the same time.By use of cRNA-based reverse-genetics system for IBDV [20], four virus mutants were recovered.Furthermore, the characteristics of recovered virus in vitro and in vivo were described and the amino acids responsible for virulence.In this paper we report the discovery that residues of 783, 862, 921 of VP3 are associated with virulence of IBDV.

Virus and Cells
The very virulent strain Harbin-1 was kindly given by Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.Harbin-1 causes 100% morbidity and mortality of specific-pathogen-free (SPF) chickens, the mean infection lethal dose (ILD 50 ) for SPF embryo is 10 -4 /0.2 ml.Primary bursal cells were derived from 18-day-old embryonated SPF eggs (Merial, Beijing, China) and were grown in Dulbecco's minimal essential medium (DMEM, Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS) and maintained with DMEM with 5% FBS [22]

Site-Directed Mutagenesis
Mutations were introduced into the cDNA of segment A of Harbin-1 according to the manufacture's instruction of QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) with minor modification.Amino acid residues 253, 284, 783, 862, 921 were located in large open reading frame of segment A and their

Transcription and Transfection of Synthetic RNAs
The experiment was performed by the protocol described by Mundt with minor alterations [24].For transcription in vitro, non-mutation and mutated plasmids of segment A and intact segment B were linearized by cleavage with XbaI and XholI respectively.After restrictive digestion, the products were adjusted to 0.5% SDS and incubated with proteinase K (0.5 mg/ml) for 1 hr at 37 .The linearized DNA templates were reco ℃ vered by ethanol precipitation, and 1 μg linearized DNA was used for transcription.Segment A and segment B was transcribed respectively.Transcription reaction mixture (30 µl) containing 40 mM Tris-HCl (pH 7.9), 10 mM NaCl, 6 mM MgCl 2 , 2 mM spermidine, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM UTP, 0.1 mM GTP, 0.25 mM cap analog [m7G(5′)ppp(5′)G] (Promega), 20 units RNasin, 130 units T7 RNA polymerase (Promega), and incubated at 37 for 1 hr.As controls, the transcri ℃ ption products were treated with either DNase or RNase (Promega).
After primary bursal cells were grown to 80% confluency in 35-mm dishes, the cells were washed with DMEM (free serum) and incubated at 37 for 10 mi ℃ nutes in a CO 2 incubator.The process was repeated again.Simultaneously, 60 μl DMEM (free serum) was incubated with 6 μl of Lipofectin reagent (Invitrogen, Carlsbad, CA, USA) for 60 min in a polystyrene tube at room temperature to form Lipofectin-DMEM mixture.Synthetic RNA transcripts of both segments resuspended in 30 μl of DEPC treated water were mixed and added to the DMEM-Lipofectin mixture, mixed gently and incubated on ice for 5 min.After removing the DMEM from the monolayers in the 35-mm dishes and replacing it with fresh 800 μl of DMEM, the nucleic acid-containing mixture was added drop-wise to the cells and swirled gently.After 2 hours of incubation at 37 , the mixture ℃ was replaced with DMEM containing 5% FCS (without rinsing the cells), and further incubated at 37 for d ℃ esired time intervals.

Virus Recovery from cRNA and Detect the Presence of Virus by AC-ELISA, RT-PCR and Plaque Assay
Two days after transfection, cells were frozen -thawed and centrifuged at 700 g to remove cellular debris.The supernatant was passaged for 4 times in the primary bursal cells, harvesting the cells for ELISA.In order to screen the recombinant virus from many samples AC-ELISA was performed.Each well of 96-wells polystyrene ELISA plates (Costar, Cambridge, MA, USA) were coated with 100 μl of chicken polyclonal IBDV antiserum, diluted in PBS at a ratio of 1:4000.After incubation at 37 for 1 hour, the plate was washed three times with ℃ washing buffer (1% Tween 80 in PBS) and each well was blocked by 100 μl of blocking buffer (0.5% gelatin in PBS) at 37 for 0.5 h.After three washes of the plate ℃ with washing buffer, 100 μl sample including positive and negative control was added in duplicate.The plate was then incubated at room temperature for 1 h and washed with washing buffer before 50 μl of MAbs M6 or B29 [25,26]., diluted 1:2500 and 1:1000 in antibody diluent (5% NaCl and 4% BSA in washing buffer) respectively, were added to the wells in duplicate.After incubation for 1 h at room temperature, the plate was washed three times with washing buffer.Subsequently, 50 μl of goat anti-mouse IgG-horseradish peroxidase (Sigma) diluted 1:1000 with antibody diluent was added.One hour later at room temperature, the plate was washed three times with washing buffer.After addition 100 μl TMB peroxidase substrate (Kirkegaard and Perry Laboratories Inc., Gaithersburg, MD, USA) and incubated at 37 for 15 min, the reaction was stopped by ℃ adding 100 μl 1 M H 3 PO 4 .The result was read by an ELISA reader at the optical density at 450 nm (OD450).If OD value of sample is greater than mean OD value plus 3 times standard deviation of negative control sample, then the sample is considered as positive and was stocked at −86 for future use.
℃ The titre of virus mutants was determined using plaque assay [27] and prepared for future animal experiment.The titre is represented as PFU/ml.

Genetic Stability Analysis
If changes in the amino acid sequence occurred during passaging viral RNA of IBDV before challenge, the identity of virus have to be confirmed.The virus mutants were subjected to RT-PCR using oligonucleotides outside 1 and outside 2 for IBDV with VP2 mutation, VP3 outupper and VP3 outlower for IBDV with VP3 muatation before challenge (Table 2).Nested PCR was ampli-fied with 783 inupper and 62, 21 inlower to identify virus with VP3 alteration (Table 2).Cloned PCR fragments of IBDV mutants were sequenced and obtained sequences were analyzed with DNAStar.

Virulence of IBDV Mutants in Young SPF Chickens
Forty eight 4-week-old SPF White Leghorn chickens were divided randomly into six groups including positive control group.Chickens were infected via eye and nose drop with total 1200 PFU .Non-inoculated hatchmates were used as negative controls.During the course of the experiment animals were observed daily for clinical signs and mortality.At 4 days p.i., all alive chickens from each group were bled and euthanized.The bursa of each chicken (include alive and dead) was removed, weighed and subdivided into two parts.One part was used for detecting the presence of IBD viral antigen by means of an AC-ELISA and RT-PCR.The second part was fixed in 10% neutral-buffered formalin for histology.Formalin-fixed bursal samples were embedded in paraffin, sectioned and stained with haematoxylin and eosin (H&E).Microscopic bursal lesion score (BLS) was determined by histopathological analysis of the bursa.BLS was evaluated on a scale of 0 to 5 as follows: 0, no abnormalities; 1, 1-20%; 2, 21-40%; 3, 41-60%; 4, 61-80%; and 5, 81-100% lymphocyte depletion [28].

Detection of Viral Antigen in Bursae after Challenge
Bursae were homogenized with homogenizer.The presence of virus in the bursal homogenate was detected with AC-ELISA which incorporated Mab 6 recognizing VP2-located epitopes [25].

Determination of Nucleotide Sequence of Harbin-1 Mutant
To establish a reverse genetics system the complete genomic sequence of Harbin-1 mutants was determined.The mutagenized plasmids were obtained with the alteration of two amino acids, Q253H-A284T, H783Q-V862M, V862M-I921V, H783Q-I921V.

Rescue of Recombinant Virus from cDNA
Primary bursal cells were transfected with synthesized cRNA of mutated segment A and intact segment B by means of lipofectin (Invitrogen).After every transfection, the resultant supernatant was used for RT-PCR and AC-ELISA to detect the presence of viruses.The samples were performed to RT-PCR after IBDV antigen was de-tectable using AC-ELISA.Electrophoresis result showed that there is one 209 bp band, whose sequence located in VP2 hypervarible region, on 1.2% agarose gel.The result of RT-PCR and AC-ELISA demonstrated that virus mutants were successfully recovered.From 10 transfection samples we obtained four mutant viruses designated as H 253/284 , H 783/862 , H 862/921 , H 921/783 and rescued Harbin-1 named rHarbin-1.

Genetic Stability Analysis
Sequence analysis of the RT-PCR products confirmed the identity of the IBDV used.No amino acid substitutions compared to the sequence of the used plasmids (p253/284 m, p783/862 m, p861/921 m, p921/783 m) were found within the region flanked by primers used for RT-PCR, proving the genetic stability of the virus during virus pass aging.

Virulence Determinants for VP2 and VP3 in Chinese vvIBDV Strain
To evaluate the virulence of all virus mutants animal experiments were performed.Animals infected with vvIBDV (rHarbin-1) and H 783/862 showed severe clinical signs of IBD.The mortality rates were 7/8 for rHarbin-

DISCUSSION
In recent years, many investigators have shown that mutations in the viral genome often lead to changes in the virulence, pathogenesis of animal viruses.A single amino acid substitution in the West Nile Virus Nonstructural protein NS2A disables its ability to inhibit Alpha/Beta interferon induction and attenuates virus in mice [18]; point mutations in an infectious bovine viral diarrhoea virus type2 cDNA transcript yields an attenu-   ated and protective viral progeny.Virulence of swine vesicular disease virus is determined at two amino acids in capsid protein VP1 and 2A protease [14].Above mentioned phenomena elicit researchers on IBDV and they dedicated to study the virulence mechanism.A number of researchers such as Brandt, Yamaguchi, Lim, Mundt and so forth assumed position 253, 279, 284 amino acids in VP2 hypervarible region control phenotype, and could bind with B lymphocyte [3,17,21,29].Lots of evidence showed that hypervarible region in VP2 involved in conformation dependent epitope and stimulate the chicken to produce protective neutralizing antibody [10,29].
The result of chickens challenged with viruses showed that H 253/284 could induced slighter lesion than parental virus vvIBDV (Harbin-1), but in H 783/862 group, there are two kinds of appearance ,the bursa in alive chickens had not showed pathological sign, which could be due to the individual difference, but the bursae of dead chickens showed severe necrosis and atrophy, B lymphocyte depletion was up to the same 80-90% as Harbin-1; in H 862/921 group, a bursa of dead chicken had the same pathological lesion as Harbin-1, lymphocyte depletion up to above 90%, in other bursae of alive chickens depletion is only 10-20%, and appear partly necrosis and atrophy; in H 921/783 group bursae had very slightly pathological lesion except minor widening interstice, suggesting bursa was slight swollen.Therefore, H 921/783 virus appeared the slightest pathological lesion among all virus mutants.Compared with mDT-VP3C and mDCT-VP3C rescued by Boot who substituted the Cterminal part of VP3 of serotype 1 vvIBDV (isolate D6948) for the corresponding part of serotype 2 IBDV [22], H 921/783 induced slighter pathological lesion than mDT-VP3C and mDCT-VP3C.mDT-VP3C and mDCT-VP3C could induced same bursa lesion as wild type D6948 and rD6948, suggesting mDT-VP3C and mDCT-VP3C had stronger residential virulence, but H 921/783 virus hardly has no residential virulence.
Our experiment demonstrated that VP3 and VP2 contain the determinant for virulence too besides VP2 in one strain.However, up to now most researches assume VP2 play an important role in virulence.The reason for this paradox about virulence controlling mechanism is unknown.Molecular determinant of virulence may depend the strains used.In addition we used two alterations of amino acid in this paper.Single alterations of aa 783, 862 and 921 were not tested, further study may be necessary to identify if single amino acid function or both of them function in virulence at the same time.

CONCLUSIONS
V862, I921 in VP3 is obvious virulence marker however I921 has more potential ability to control virulence than V862 and H783.Through animal challenge test we make clear the site in VP2 and VP3 involved in virulence, furthermore, the ability of 862 and 921 to control virulence in VP3 is more powerful than 253 and 284 in VP2.
of 4-week-old SPF chickens were infected via the eye and nose drop; ** BLS of of each chicken investigated.Values within the same row with the same superscript letters are not significant (P < 0.05).

Table 1 .
Different AA in VP3 for Harbin-1 and vaccine strain, serotype II strain.

Table 2 .
Oligonucleotides used for amplification of Harbin-1 sequence*.Sequence and location of the oligonucleotide used in the study.Underlined nucleotides are virus-specific.Altered nucleotides for mutagenesis are in lowercase, the altered coding nucleotide triplets are highlighted in boldface.Used restriction sites are highlighted in boldface and appropriate restriction enzymes are named.The positions where the primers bind (nucleotide number) are in accordance with the sequence of strain P2(Mundt et al., 1995).

Table 3 .
Results of chicken challenged by four recombinant viruses.