Sequence analysis of the variable VP2 gene of infectious bursal disease viruses passaged in Vero cells.

Korean field infectious bursal disease viruses (IBDVs) were isolated from IBDV suspected commercial chickens. A previous study revealed that these IBDV field isolates were virulent or very virulent IBDVs. The isolates were passaged three times in the chorioallantoic membrane of specific-pathogen-free embryonated chicken eggs and four times in Vero cells. After passage, viral RNAs were isolated and purified to determine the genetic changes. The hypervariable regions of the VP2 gene were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). To confirm the genetic changes, PCR products were cloned, sequenced and compared to the sequences of the parental IBDVs and published IBDV strains. By sequencing analysis, the passaged IBDVs had amino acid changes at positions 253 (Q --> H), 279 (D or N --> N) and 284 (A --> T) which were commonly found in the attenuated IBDV strains. Two serines in the serine-rich heptapeptide (residue 326-332) were substituted into other amino acids which were similar to the IBDV vaccine strains.

Protection against very virulent infectious bursal disease virus in chickens immunized with DNA vaccines.

Plasmid DNA vaccines pcDNA-VP2 expressing only VP2 protein and pcDNA-VP243 expressing VP2, VP4 and VP3 proteins of very virulent infectious bursal disease virus (vvIBDV) Korean SH/92 strain were constructed. The expression of viral proteins from constructed DNA vaccines was confirmed by an in vitro transcription/translation system and transfection in COS-7 cells. To investigate the protective efficacy of these DNA vaccines, 2-week-old chickens were injected intramuscularly and intraperitoneally with pcDNA-VP2 and pcDNA-VP243 twice at 2-week intervals. On week 2 after the second immunization, chickens were orally challenged with the vvIBDV SH/92 strain and observed for 10 days. Antibodies specific to IBDV were not detected by enzyme-linked immunosorbent assay in DNA vaccination groups before challenge but were induced after challenge. The immunized groups exhibited a higher survival rate and lower bursal atrophy as compared with the non-immunized groups after challenge. The survival rates of pcDNA-VP243 and pcDNA-VP2 groups were 70 and 50%, respectively, but the survival rate of challenge control group was only 10%. In the ConA-induced lymphocyte proliferation assay of peripheral blood and splenic lymphocytes, the immunized groups showed significantly higher proliferation responses (P< 0.05) than non-immunized groups. The maintenance of T cell proliferation activity in DNA vaccination groups may be closely related to the protection against vvIBDV. These results suggest that our plasmid DNA vaccines induced high protective immunity against vvIBDV, in which cell mediated immune response rather than humoral immune response seemed to contribute to the protection of chickens against vvIBDV infection.

Partial sequence of the spike glycoprotein gene of transmissible gastroenteritis viruses isolated in Korea.

The spike (S) glycoprotein of transmissible gastroenteritis virus (TGEV) is the predominant inducer of neutralizing antibodies and has been implicated in virulence and host cell tropism. In this study, the nucleotide and deduced amino acid sequences of the amino terminal half of the S glycoprotein gene of one Korean field TGEV strain (133) isolated in 1997 and three Korean field TGEV strains (KT2, KT3 and KT4) isolated in 2000 and HKT2 strain, KT2 passaged 104 times in ST cells, were determined. The amino terminal half of the S glycoprotein gene including antigenic sites A, B, C and D, were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). Amplified PCR products were cloned, sequenced, and compared with published sequences for non-Korean TGEV strains. Korea TGEV field strains had 98.5-99.5% nucleotide sequence and 97.2-99.0% amino acid sequence similarity with each other. They had 96.5-99.0% nucleotide sequence similarity and 94.9-97.6% amino acid sequence similarity compared to non-Korean TGEV strains. Korean TGEV strains had several specific nucleotide and amino acid sequences which were not found in foreign TGEV or PRCV strains. HKT2 strain differed by 0.89% in nucleotide and 2.03% amino acid sequences compared to original KT2 strain although the regions forming four antigenic sites were not changed. By phylogenetic tree analysis, Korean field TGEV strains were branched into different groups from non-Korean TGEV or PRCV strains. Korean TGEV field strains KT2 and 133 were branched in separate groups that were differentiated from the other Korean TGEV strains. The Korean TGEV strains seemed to be evolved from a separate lineage of TGEV strain.