Fig. S1. HA Epitope Expression from Cdna Constructs. DF-1 Cells Were Transfected With

Fig. S1. HA epitope expression from cDNA constructs. DF-1 cells were transfected with each ha-containing cDNA construct or VP1/pcDNA as indicated. After 48hr, cells were fixed by acetone and subjected to immunofluoresent staining with anti-HA tag monoclonal antibody (Covance, Emeryville, CA, USA). HA epitope expression was detected in every cDNA construct (c-g) except for SegA-HA5 (a). But the expression of HA tag was revealed when co-expressing VP1 in transfected cells (b). There was no fluorescence from cells with mock-transfection (data not shown).The scale bar on each photo represents 20 µm.

Fig. S2. Different HA-IBDVs produced various plaque sizes.Plaques formed by different ha-containing cDNA clones showed various plaque sizes.

Fig. S3. HA tag-labeled viral protein by IFA. DF-1 cells were infected with HA-IBDV P3 stock (MOI=1). HA4+1HA was not included in this experiment since a MOI=1 could not be achieved due to the lower viral titer. After incubation for 24hr, IFA was followed as described previously with anti-HA mAb (Covance, Emeryville, CA, USA) to reveal expression location in infected cells (20x). No fluorescence was observed in SegA+SegB infected cells (data not shown). The scale bar on each photo represents 10 µm.

Fig. S4. Western blotting for HA-IBDVs. DF-1 cells were infected with HA-IBDVs P2 stock at MOI=0.2. After 22 hr, cells were washed three times with sterile phosphate buffered saline (SPBS) and cellular proteins were extracted by CytoBuster™ Protein Extraction Reagent (Novagen/EMD, La Jolla, CA, USA) and subjected to SDS-PAGE in 15% acrylamide gel, followed by Western blotting with anti-HA monoclonal antibody (Covance, Emeryville, CA, USA). The stained protein bands were revealed by 3,3',5,5'-tetramethylbenzidine (TMB) membrane peroxidase substrate (KPL). HA-VP5 showed a molecular weight around 20 kDa; HA-VP4 was about 30 kDa and VP1-HA had a band around 90 kDa. However, there was a protein band of about 38 kDa size of unknown identify in HA5+B and HA5+1HA infected cells. Based on the molecular weight, it could be a dimer formed by HA-VP5 fusion protein.

Fig. S5. Negative staining of HA-IBDVs by electron microscopy. HA-expressing IBDVs all have similar viral particle morphology and size to the RG-IBDV.

Fig. S6. Bursal weight/body weight (B/B) ratio for HA-IBDV infected chickens. Two-week-old SPF chickens were orally inoculated with 1 x 104 pfuof RG-IBDV (A+B), HA5-IBDV, HA4-IBDV or 1HA-IBDV and boosted at 4-week-old with 1 x 105 pfu of virus. After 24 days post the first infection (DPI), bursae and chickens were weighted for calculation of bursa weight/body weight ratio (B/B ratio) using formula (bursal weight)/(body weight) x 1000. Lower number of B/B ratio indicates bursal atrophy. There was no significant difference between each group (p>0.05).

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