Three classic IBDV strains were previously isolated from commercial layer chicken flocks and shown to be phylogenetically related to vaccine strains but pathogenic in susceptible chickens. In this study, their viral genomes were sequenced and compared to sequences of vaccines being used in those flocks. The vaccine strains examined were sequenced directly from the manufacturer and had identical genome segment B sequences. Compared to these vaccines, the GA-1, H-30 and CS-2-35 isolates each had one silent mutation in the gene that encodes VP1. Compared to the two vaccines used at the time CS-2-35 was isolated, the segment A sequence of CS-2-35 contained numerous nucleotide and amino acid mutations suggesting the CS-2-35 virus was not closely related to these vaccines. This virus however did have amino acid mutations in VP2 that are reported to be necessary for replication in cell culture and lacked two of the three amino acid mutations previously shown to be necessary for virulence. These data suggest that CS-2-35 was a descendant from an attenuated strain of IBDV. When the segment A genomic sequences of the GA-1 and H-30 viruses were compared to the vaccines being used in those flocks they were most closely related to the attenuated D78 vaccine strain. In genome segment A, three nucleotide mutations in GA-1 and four in H-30 were observed compared to the D78 classic vaccine. These nucleotide mutations caused one amino acid (H253N) change in the GA-1 virus and two amino acids (H253Q and G259D) were different in the H-30 virus. In addition, both the GA-1 and H-30 viruses had the amino acid G76 in VP2 that appears to be unique to the vaccine D78. The data suggest that GA-1 and H-30 are genetically related and have a common ancestor even though they were isolated from geographically distant flocks. The evidence also suggests that GA-1, H-30 and CS-2-35 could be reversions from attenuated vaccine viruses or by coincidence genetically resemble classic IBDV vaccines. It should be noted that some of the classic virus vaccines were not being used according to the manufacturer's recommendations at the time the GA-1, H-30 and CS-2-35 strains were isolated. Together, the molecular and pathogenicity data indicate that a single amino acid mutation from Histidine (H) to Glutamine (Q) or Asparagine (N) at position 253 in VP2 will markedly increase the virulence of an attenuated IBDV.

Twenty bursal samples were obtained from four infectious bursal disease virus (IBDV)-vaccinated layer flocks experiencing problems with immune suppression that was thought to be infectious bursal disease. All the samples were found to be positive for IBDV by reverse transcriptase polymerase chain reaction (RT-PCR). Restriction fragment length polymorphism analysis of the samples identified them as classic molecular group 3 and group 4 viruses. Two samples from each of the four flocks were sequenced, and within a flock, these sequences were identical; however, between flocks, some differences were observed. One virus from each of the four flocks was selected for further analysis. The VP2 hypervariable sequence region of samples GA-1, H-30, and CS-2-35 had nucleotide and amino acid similarities with the D78 and Vi Bursa G classic vaccines that were used in those flocks. The sequence of HPR-2 was similar to the Bursa Vac 4 vaccine used in that flock and the STC virulent classic IBDV strain. The deduced amino acid sequence of these isolates revealed that all the isolates had proline at position 222, which is characteristic of U.S. classic viruses. The phylogenetic analysis of these isolates on the basis of the VP2 hypervariable amino acid sequence clustered GA-1, H-30, and CS-2-35 with the D78 vaccine and HPR-2 with STC. The pathogenicity of these isolates was tested in specific-pathogen-free chickens. Bursa-body weight (B-BW) ratios and histopathologic lesion scores in the bursa were determined. Gross lesions were observed in the bursa, and the B-BW ratios of the birds infected with all four wild-type viruses were significantly different compared with the D78 vaccine and uninoculated control groups. Histopathology of the bursa from groups infected with GA-1, H-30, CS-2-35, and HPR-2 showed different degrees of follicular depletion and necrosis. A very slight lymphoid depletion was observed in the D78-infected group at 5 days postinoculation, and no microscopic lesions were observed in this group at 8 days postinoculation or at any time in the uninoculated control group. The bursa collected from the field virus and D78-infected birds at necropsy revealed the presence of IBDV via RT-PCR, and the VP-2 hypervariable nucleotide sequences of the GA-1, H-30, CS-2-35, HPR-2, and D78 samples were identical to the original viral isolates and vaccine, respectively.

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