Antibodies to infectious bronchitis virus (IBV) cross-react with turkey coronavirus (TCV) in immunofluorescence assay (IFA) indicating that IBV and TCV may share an amino acid sequence similarity. To determine its extent, the gene encoding the nucleocapsid (N) protein of TCV was amplified by reverse transcription-PCR (RT-PCR) from RNA purified from intestines of embryos of turkeys infected with various TCV isolates and from allantoic fluid of chicken embryos infected with IBV M41 strain, the obtained N genes were cloned, sequenced and compared with known sequences of N genes of five IBV strains. The primers for amplification were designed from the genome of IBV PCR products were obtained only from two of eight TCV isolates tested. It was found that the two TCV isolates were identical with five IBV strains by 90.1-94.1% at the N gene level. It was also observed that the N gene of eight TCV isolates originating from various regions of the USA could not be amplified by the primers designed from the N gene of bovine coronavirus (BCV).

Five hammerhead-type ribozymes were designed and cloned to cleave infectious bursal disease virus (IBDV) RNA and inhibit protein synthesis from cloned full-length viral cDNA genes. Two ribozymes (R1 and R2) were directed to the large viral RNA segment gene and three ribozymes (R3, R4, and R5) were directed to the small viral RNA segment gene. Targets for the ribozymes were produced from cloned full-length coding regions of both small and large viral RNA segment genes. Ribozymes and their corresponding targets were synthesized as in vitro transcripts. Despite several attempts at different temperatures, no cleavage of viral RNA transcripts with four of the ribozymes (R1, R2, R3, and R5) was observed. One of the ribozymes, R4, was effective in cleaving the viral RNA polymerase gene transcripts in a magnesium-dependent manner. Ribozyme R4 caused 82% reduction in the synthesis of the viral RNA polymerase gene product. The inhibition was specific since there was no change in the rate of synthesis of the Escherichia coli beta-galactosidase protein. The results suggest that ribozyme R4 can be used as potential anti-IBDV agent. It was also demonstrated that the hammerhead-type ribozymes can cleave sites other than conventional GUC sequence motif as the cleavage site of ribozyme R4 had GUU motif which conformed to the NUX consensus motif.

Improved methods of reverse transcription, polymerase chain reaction (PCR) amplification, and cloning of full-length coding region of both strands of infectious bursal disease virus (IBDV) variant strain E genome were developed. Denaturation of IBDV RNA by heat in the presence of primers and use of a reverse transcriptase lacking RNase-H activity produced full-length coding region and partial non-coding region cDNA copies of the viral genomic segments. Digestion of the RNA component of RNA-cDNA hybrids by RNase-H followed by long and accurate PCR (LA-PCR) amplification of IBDV cDNA in a single step resulted in the synthesis of 3182 base-pairs (bp) of segment A and 2777 bp of segment B cDNA copies of IBDV genome. The resulting amplicons were successfully cloned and sequenced revealing their identity of IBDV. The LA-PCR method can be utilized for the amplification and cloning of the other IBDV strains or isolates and will greatly enhance the availability of sequence information or infectious cDNA copies of IBDV.

Infectious bursal disease virus (IBDV), a member of the birnaviridae family, contains a bisegmented double-stranded RNA (dsRNA) genome. The segments are linked covalently at 5′ termini by a large (90 kDa) viral genomic protein that migrates similar to viral RNA dependent RNA polymerase of IBDV. A proteinase K digestion based approach and acid-guanidium-phenol-chloroform (AGPC) RNA extraction method were used to extract dsRNA of IBDV from infected bursae. After extraction, dsRNA of IBDV was purified by precipitation with lithium chloride. The yield and purity of dsRNA of IBDV extracted by AGPC method was lower than that of proteinase K digestion based approach. This observation correlates with the presence of a genome-linked protein in IBDV. Although dsRNA obtained by both methods are suitable for reverse transcription-polymerase chain reaction (RT-PCR) amplification of at least up to 1201 base pairs (bp) of cDNA, dsRNA extracted by the proteinase K digestion method is more suitable than that by AGPC method for the amplification of longer fragments (1958 bp) of IBDV cDNA by PCR.

A polymerase chain reaction (PCR)-based method to measure complementary DNA (cDNA) and RNA levels of infectious bursal disease virus (IBDV) was developed. Quantification was achieved by quantitative competitive PCR (QC-PCR) amplification. A competitor, a deletion mutant of the wild type IBDV cDNA, was 10-fold serially diluted and co-amplified with IBDV cDNA after being reversely transcribed from the viral RNA. After agarose gel electrophoresis, staining, and densitometric scanning, the bands on the digitized images were analyzed and quantified by computer-assisted image analysis. Complementary DNA of standard, as well as variant strains, of serotype 1 IBDV was detected and quantified using the same QC-PCR procedures. The assay could measure IBDV cDNA levels ranging from 1 microgram to 45 fg and RNA levels ranging from 9 micrograms to 45 fg. The results indicated that QC-PCR is sensitive, easy to perform, and suitable for routine quantitation of IBDV cDNA or RNA levels.

A polymerase chain reaction (PCR)-generated digoxigenin-labeled nonradioactive oligonucleotide probe was developed and utilized in slot-blot hybridization coupled with chemiluminescence for the detection of infectious bursal disease virus (IBDV). The probe was prepared from the RNA of the standard challenge strain (STC) of IBDV serotype 1 by reverse transcription followed by 2 PCR amplifications with 2 separate sets of primers. RNA of STC viruses prepared from bursae infected with STC viruses was subjected to the first PCR with the outer primers V8 and V9 that amplified a 607-base pair (bp) segment. The PCR product from the first PCR was eluted following agarose gel electrophoresis and subjected to the second PCR with the nested primers V6 and V7 that flanked a 351-bp segment. In the second PCR, dTTP was substituted by digoxigenin-11-dUTP in the PCR reaction mixture so that the amplified 351-bp DNA products were labeled with digoxigenin. The specificity of the PCR-generated digoxigenin-labeled probe was tested on different strains of IBDV, several unrelated avian viruses, and bacteria by slot-blot hybridization assay. Hybridization was detected by chemiluminescence. The sensitivity of the probe was assayed using lo-fold serial dilutions of purified RNA from the STC strain of IBDV. The PCR-generated digoxigenin-labeled probe hybridized with genomic RNA of STC and variant strains A, D, E, G, and GLS-5 of IBDV serotype 1 but not OH strain of IBDV serotype 2. The probe did not react with avian reovirus, infectious bronchitis virus, Salmonella enteritidis, Escherichia coli, or Staphylococcus aureus. The probe was very sensitive, and as little as 72 fg of RNA from the STC strain of IBDV could be detected. The results indicate that this PCR-generated digoxigenin-labeled nonisotopic probe is specific for IBDV and may be utilized in a diagnostic assay for all IBDV serotype 1 strains.