Differential detection of infectious bursal disease virus serotypes, using cDNA probes to VP2 coding region

Frederick S. B. Kibenge From the Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, 550 University Ave, Charlottetown, Prince Edward Island, Canada, C1A 4P3.

Search for other papers by Frederick S. B. Kibenge in
Current site
Google Scholar
PubMed
Close
 BVM, PhD

Click on author name to view affiliation information

Summary

Two nonoverlapping clones, pOH405 and pOH632, containing cdna inserts in the VP2 coding region of genome segment A were selected from a cdna library prepared from the double-stranded rna genome of the OH strain of infectious bursal disease virus (ibdv) of serotype 2. Clone pOH405, which is located in the hypervariable segment of VP2, is 328 base pairs long, has nucleotide sequence homology of 72 to 73%, and amino acid sequence homology of 64 to 67% with ibdv strains of serotype 1. Clone pOH632, which is located in the highly conserved C-terminal part of VP2, is 230 base pairs long, has nucleotide sequence homology of 87 to 88%, and amino acid sequence homology of 100% with ibdv serotype 1. The lower detection limit of 32P-labeled probes prepared from both clones was 10 ng of OH-ibdv double-stranded rna, using high-stringency conditions of hybridization (54 C, 50% formamide) and washing (55 C, 0.015M NaCl, 0.0015M trisodium citrate, pH 7.0, with 0.1% sodium dodecyl sulfate), and autoradiography for 24 hours. Under these conditions, the dot-blot hybridization assay for detection of serotype 2 ibdv double-stranded rna was 1,000 times more sensitive, using probe pOH632, but only 10 times more sensitive, using probe pOH405, compared with the assay for ibdv serotype 1, using the same probes. Thus, probe pOH632 could differentiate between the 2 ibdv serotypes by nucleic acid hybridization.

Summary

Two nonoverlapping clones, pOH405 and pOH632, containing cdna inserts in the VP2 coding region of genome segment A were selected from a cdna library prepared from the double-stranded rna genome of the OH strain of infectious bursal disease virus (ibdv) of serotype 2. Clone pOH405, which is located in the hypervariable segment of VP2, is 328 base pairs long, has nucleotide sequence homology of 72 to 73%, and amino acid sequence homology of 64 to 67% with ibdv strains of serotype 1. Clone pOH632, which is located in the highly conserved C-terminal part of VP2, is 230 base pairs long, has nucleotide sequence homology of 87 to 88%, and amino acid sequence homology of 100% with ibdv serotype 1. The lower detection limit of 32P-labeled probes prepared from both clones was 10 ng of OH-ibdv double-stranded rna, using high-stringency conditions of hybridization (54 C, 50% formamide) and washing (55 C, 0.015M NaCl, 0.0015M trisodium citrate, pH 7.0, with 0.1% sodium dodecyl sulfate), and autoradiography for 24 hours. Under these conditions, the dot-blot hybridization assay for detection of serotype 2 ibdv double-stranded rna was 1,000 times more sensitive, using probe pOH632, but only 10 times more sensitive, using probe pOH405, compared with the assay for ibdv serotype 1, using the same probes. Thus, probe pOH632 could differentiate between the 2 ibdv serotypes by nucleic acid hybridization.

All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 24 24 6
PDF Downloads 22 22 5
Advertisement