Development and validation of a real-time Taqman PCR assay for the detection and quantitation of infectious laryngotracheitis virus in poultry.

In this study, the development and validation of a real-time (ReTi) PCR assay is described using a Taqman labeled probe for the detection and quantitation of infectious larygotracheitis virus (ILTV) in chickens. The ReTi ILTV assay was highly specific with a quantitation limit of 100 viral template copies per amplification reaction. In experimentally infected, birds during early acute stages of infection, an average of 6.67 log(10) viral template copies/amplification reaction were detected, while at chronic late stages of infection an average of 2.86-3.27 log(10) viral template copies/amplification reaction were detected. A total of 246 tracheal swab samples collected from natural outbreaks of the disease were tested by virus isolation and the ReTi ILTV assay. Both assays agreed in 37% of the samples tested and the ReTi ILTV assay detected approximately 3.7 times more positives samples than virus isolation. A minimum of 5 log(10) viral template copies/amplification reaction were required from a tracheal swab to render a virus isolation positive result. In conclusion, the ReTi ILTV assay was highly specific, sensitive, reproducible, and capable of reliably quantifying viral nucleic acid directly from clinical samples.

Development and validation of a real-time Taqman polymerase chain reaction assay for the detection of Mycoplasma gallisepticum in naturally infected birds.

In this study, we report the development and validation of a real-time polymerase chain reaction (PCR) assay using a Taqman-labeled probe for the detection of Mycoplasma gallisepticum (MGLP assay). The MGLP assay was highly specific with a detection limit of 25 template copies per reaction and a quantification limit of 100 template copies per reaction. Validation of the assay was completed with 1247 samples (palatine cleft and tracheal swabs) from M. gallisepticum-positive and -negative chicken flocks. The MGLP assay was compared to an enzyme-linked immunosorbent assay (ELISA), a conventional polymerase chain reaction assay (mgc2 PCR), and isolation of M. gallisepticum from naturally infected flocks. A total of 805 samples collected from negative flocks, as verified by ELISA and/or mgc2 PCR, were negative by the MGLP assay. A total of 442 samples were collected from positive flocks, of which a total of 228 samples were positive by the MGLP assay. These results agreed for 98.87% of the samples when tested by mgc2 PCR. When comparing the MGLP assay with M gallisepticum isolation, the MGLP assay was more sensitive than isolation for detecting positive birds from a positive flock, 172/265 and 50/265, respectively. Overall, the MGLP assay and M. gallisepticum isolation agreed for 52.8% of the samples tested. In conclusion, the MGLP assay was highly specific, sensitive, and reproducible, and allowed the quantification of template copies directly from clinical samples.

Rapid differentiation of avian infectious bronchitis virus isolates by sample to residual ratio quantitation using real-time reverse transcriptase-polymerase chain reaction.

A rapid diagnostic assay for differentiating avian infectious bronchitis virus (IBV) isolates was developed. The basis of the assay is the cleavage of target RNA by RNase H mediated by sequence-specific chimeric oligonucleotides followed by sample to residual ratio quantitation (SRRQ) using RRT-PCR. Four serotype-specific chimeric oligonucleotides were designed, one each for the Massachusetts, Connecticut, Arkansas, and Delaware/Georgia 98 serotypes, and tested for their ability to mediate specific cleavage of target RNA from known homologous and heterologous strains of IBV. Specific cleavage of target RNAs by each chimeric oligonucleotide was verified using agarose gel analysis and RRT-PCR. There were no non-specific cleavage products. Eight different IBV strains representing seven serotypes were tested and each chimeric oligonucleotide mediated cleavage of target RNA only from strains within the serotype that the chimeric was designed against. The SRRQ assay was evaluated on 15 samples without prior knowledge of their grouping and correctly identified the serotype of each sample. The assay is rapid; six samples can be tested in approximately 4 h. In addition, the primer set amplifies all IBV RNAs tested to date and provides a built in control for detecting IBV whether it is typeable or not.

Detection of infectious bronchitis virus by real-time reverse transcriptase-polymerase chain reaction and identification of a quasispecies in the Beaudette strain.

In this report, we describe a real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) diagnostic test for infectious bronchitis virus (IBV) with the use of fluorescence resonance energy transfer (FRET) technology. Two primers that amplify a 383-base pair product between nucleotide positions 703 and 1086 relative to the start codon for the S1 gene of the Massachusetts 41 virus were designed and used to amplify the Beaudette, Massachusetts 41, Florida 18288, Connecticut, Iowa 97, Arkansas DPI, CA/NE95/99, DE/072/ 92, and GA/0470/98 strains of IBV. The primers were specific and did not amplify New Castle disease virus, Mycoplasma spp., or infectious laryngotracheitis virus. For RRT-PCR by FRET, an anchor probe conjugated to fluorescein and a detection probe conjugated to a red fluorophore were designed to anneal to a hypervariable region within the 383-base pair product. The level of sensitivity was 1 x 10(4) RNA molecules used as starting template. After amplification, a melting curve analysis was conducted to specifically identify IBV types. Because of sequence differences in the annealing position of the detection probe, the Arkansas, Connecticut, Beaudette, and Massachusetts 41 strains could be differentiated. No fluorescence was observed for the DE/072/ 92 and GA/0470/98 viruses with the anchor and detection probes. When the Beaudette strain was examined, two melting peaks were observed at 44 C and 51 C, indicating a quasispecies in that laboratory strain of IBV. Routine typing of vaccine strains of IBV was possible with this technology, but high standard deviations associated with the melting curve analysis of the FRET probes described herein made it difficult to use this test reliably for routine typing of IBV field isolates.

Spike glycoprotein cleavage recognition site analysis of infectious bronchitis virus.

The spike glycoprotein of infectious bronchitis virus (IBV), a coronavirus, is translated as a precursor protein (So), then cleaved into two subunits (S1 and S2) by host cell serine proteases. In this study, we compared the cleavage recognition site of 55 IBV isolates to determine if the cleavage recognition site sequence, which consists of five basic amino acid residues, correlates with host cell range, serotype, geographic origin, and pathogenicity as it does in orthomyxoviruses and paramyxoviruses. The most common cleavage recognition site observed (33 of 55 viruses) was Arg-Arg-Ser-Arg-Arg, representing at least 11 different serotypes. Thus, cleavage recognition site does not appear to correlate with serotype. We also determined that cleavage recognition site sequence does not correlate with pathogenicity because attenuated and pathogenic isolates (different passages of the same virus) contain identical cleavage recognition site sequences. In addition, nephropathogenic strains had the same cleavage recognition site sequence as many nonnephropathogenic isolates. Cleavage recognition site sequence does correlate with viruses in different geographic regions, which may be an important characteristic to examine in epidemiologic studies. An IBV monoclonal antibody neutralization-resistant mutant (NR 18) had an unusual substitution of Ile for Arg at the fourth position, giving the sequence Arg-Arg-Ser-Ile-Arg, which likely prevents cleavage and, thus, destroys the conformationally dependent monoclonal antibody binding epitope. Six residues on the amino-terminal side of the cleavage recognition site are conserved in 31% of the isolates and consist of only one or two basic amino acids. Thus, the number of basic residues around the cleavage recognition site does not appear to correlate with increased cleavability, host cell range, and increased virulence as it does with envelope glycoproteins in orthomyxoviruses and paramyxoviruses.

Molecular characterization of infectious bronchitis virus isolates foreign to the United States and comparison with United States isolates.

Eleven infectious bronchitis virus (IBV) isolates foreign to the United States were analyzed by using reverse transcriptase (RT)-polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) and S1 glycoprotein gene sequencing. Two of the isolates generated RFLP patterns that resembled the Mass 41 strain. Seven novel RFLP patterns were detected among the other nine foreign IBV isolates. Five of the foreign isolates were further analyzed by S1 glycoprotein gene sequencing in our laboratory. Phylogenetic analysis of S1 glycoprotein-deduced amino acid sequences for 4/91 pathogenic, 4/91 attenuated, and Variant 1 were greater than 90% similar to viruses belonging to the 793/B serogroup and, therefore, are possibly serologically related. Variant 2 was only 81.0% similar to viruses belonging to the European serogroup B, and, therefore, predicting its serotype is difficult. Isolates 98-07484 and 97-8123 were genotypically unique and therefore might be serologically unique. With the RFLP patterns and the deduced S1 amino acid sequence data as a reference, none of the IBV isolates foreign to the United States have been detected in the United States.

Infectious bronchitis virus S2 gene sequence variability may affect S1 subunit specific antibody binding.

The S2 gene of several strains of infectious bronchitis virus (IBV) belonging to the Arkansas, Connecticut, and Florida serotypes was sequenced. Phylogenetic analysis of the S2 gene nucleotide and deduced amino acid sequence data resulted in groups of strains that were the same as groupings observed when S1 sequence data was used. Thus, it appears that S2 subunits are conserved within a serotype but not between serotypes. Although the sequence differences were small, we found that only a few amino acid differences were responsible for different secondary structure predictions for the S2 subunit. It is likely that these changes create different interactions between the S1 and S2 subunits, which could affect the conformation of the S1 subunit where serotype specific epitopes are located. Based on this sequence data, we hypothesize that the S2 subunit can affect specific antibody binding to the S1 subunit of the IBV spike glycoprotein.

Mutations of the cathepsin C gene are responsible for Papillon-Lefèvre syndrome.

Papillon-Lefèvre syndrome (PLS) is an autosomal recessive disorder characterised by palmoplantar hyperkeratosis and severe early onset periodontitis that results in the premature loss of the primary and secondary dentitions. A major gene locus for PLS has been mapped to a 2.8 cM interval on chromosome 11q14. Correlation of physical and genetic maps of this interval indicate it includes at least 40 ESTs and six known genes including the lysosomal protease cathepsin C gene (CTSC). The CTSC message is expressed at high levels in a variety of immune cells including polymorphonuclear leucocytes, macrophages, and their precursors. By RT-PCR, we found CTSC is also expressed in epithelial regions commonly affected by PLS, including the palms, soles, knees, and oral keratinised gingiva. The 4.7 kb CTSC gene consists of two exons. Sequence analysis of CTSC from subjects affected with PLS from five consanguineous Turkish families identified four different mutations. An exon 1 nonsense mutation (856C-->T) introduces a premature stop codon at amino acid 286. Three exon 2 mutations were identified, including a single nucleotide deletion (2692delA) of codon 349 introducing a frameshift and premature termination codon, a 2 bp deletion (2673-2674delCT) that results in introduction of a stop codon at amino acid 343, and a G-->A substitution in codon 429 (2931G-->A) introducing a premature termination codon. All PLS patients were homozygous for cathepsin C mutations inherited from a common ancestor. Parents and sibs heterozygous for cathepsin C mutations do not show either the palmoplantar hyperkeratosis or severe early onset periodontitis characteristic of PLS. A more complete understanding of the functional physiology of cathepsin C carries significant implications for understanding normal and abnormal skin development and periodontal disease susceptibility.