Deborah A. Hilt

Development and evaluation of a real-time taqman RT-PCR assay for the detection of infectious bronchitis virus from infected chickens

Abstract It is important to rapidly differentiate infectious bronchitis virus (IBV) from disease agents like highly pathogenic avian influenza virus and exotic Newcastle disease virus, which can be extremely similar in the early stages of their pathogenesis. In this study, we report the development and testing of a real-time RT-PCR assay using a Taqman®-labeled probe for early and rapid detection of IBV. The assay amplifies a 143-bp product in the 5′-UTR of the IBV genome and has a limit of detection and quantification of 100 template copies per reaction. All 15 strains of IBV tested as well as two Turkey coronavirus strains were amplified, whereas none of the other pathogens examined, tested positive. Evaluation of the assay was completed with 1329 tracheal swab samples. A total of 680 samples collected from IBV antibody negative birds were negative for IBV by the real-time RT-PCR assay. We tested 229 tracheal swabs submitted to two different diagnostic laboratories and found 79.04% of the tracheal swabs positive for IBV by real-time RT-PCR, whereas only 27.51% of the samples were positive by virus isolation, which is the reference standard test. We also collected a total of 120 tracheal swabs at six different time points from birds experimentally infected with different dosages of IBV and found that, independent of the dose given, the viral load in the trachea plateau at 5 days post-inoculation. In addition, an inverse relationship between the dose of virus given and the viral load at 14 days post-inoculation was observed. Finally, we tested 300 total tracheal swab samples, from a flock of commercial broilers spray vaccinated for IBV in the field. The percentage of birds infected with the IBV vaccine at 3, 7, and 14 days post-vaccination was 58%, 65%, and 83%, respectively, indicating that only slightly more than half the birds were initially infected then the vaccine was subsequently transmitted to other birds in the flock. This observation is significant because coronaviruses, which have a high mutation rate, can revert to pathogenicity when bird-to-bird transmission occurs. The real-time RT-PCR test described herein can be used to rapidly distinguish IBV from other respiratory pathogens, which is important for control of this highly infectious virus. The test was extremely sensitive and specific, and can be used to quantitate viral genomic RNA in clinical samples.

Avian coronavirus infectious bronchitis attenuated live vaccines undergo selection of subpopulations and mutations following vaccination

Summary In this study, we were interested in determining if high titered egg adapted modified live infectious bronchitis virus (IBV) vaccines contain spike gene related quasispecies that undergo selection in chickens, following vaccination. We sequenced the spike glycoprotein of 12 IBV vaccines (5 different serotypes from 3 different manufacturers) directly from the vaccine vial, then compared that sequence with reisolated viruses from vaccinated and contact-exposed birds over time. We found differences in the S1 sequence within the same vaccine serotype from different manufacturers, differences in S1 sequence between different vaccine serials from the same manufacturer, and intra-vaccine differences or quasispecies. Comparing the sequence data of the reisolated viruses with the original vaccine virus, we were able to identify in vivo selection of viral subpopulations as well as mutations. To our knowledge, this is the first report showing selection of a more fit virus subpopulation as well as mutations associated with replication of modified live IBV vaccine viruses in chickens. This information is important for our understanding of how attenuated virus vaccines, including potential vaccines against the SARS-CoV, can ensure long-term survival of the virus and can lead to changes in pathogenesis and emergence of new viral pathogens. This information is also valuable for the development of safer modified live coronavirus vaccines.

Identification of amino acids involved in a serotype and neutralization specific epitope with in the s1 subunit of avian infectious bronchitis virus

SummaryLocalization of neutralizing, serotype specific epitopes of infectious bronchitis virus has been difficult because these epitopes are conformationally dependent. We identified amino acids involved in a serotype specific, conformationally dependent epitope by analysis of the S1 gene of 13 monoclonal antibody-neutralization-resistant mutants. Substitutions in the predicted amino acid sequence of these mutants were located at residues 304 and/or 386. Most of the substitutions at residue 304 were from threonine to isoleucine, whereas the substitutions at residue 386 were from arginine to proline, histidine, cysteine, or tryptophan. Based on this data, it appears that AA residues at 304 and 386 on the S1 glycoprotein are involved in a virus neutralizing serotype specific epitope.

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.

Data from 11 years of molecular typing infectious bronchitis virus field isolates.

Abstract In 1993, a new molecular typing method for infectious bronchitis virus (IBV) was introduced. This method uses reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) analysis of the spike gene to obtain RFLP patterns that correlate with serotype. Using that test at the Poultry Diagnostic and Research Center (PDRC, University of Georgia, Athens, GA), we have identified a total of 1523 IBV isolates in the past 11 yr. The data were obtained from clinical samples submitted to our laboratory from birds with clinical signs characteristic of IBV infection. The samples are primarily from the southeastern United States but are also from many other states as well as from outside the United States. Most of the isolations occurred during July, followed by May, April, November, October, and January. The fewest number of isolates identified on an annual basis was 20 in 2003. An unusually high number of isolations occurred in 1997 (318 isolations) and 1999 (246 isolations), which coincided with the GAV variant virus and GA98 variant virus outbreaks respectively. By far, the Ark-DPI strain was the most frequently identified type of IBV and ranged from 23% to 65% of total isolations per year. Ark-like isolates, defined as having a similar but unique RFLP pattern from the Ark-DPI vaccine strain were identified every year of the study except in 1996. In addition, new Ark-like isolates continued to emerge each year (except in the year 2000) beginning in 1997, reflecting the ability of that IBV type to undergo genetic drift. Eighty-two different variant viruses were identified although only two (GAV and GA98) became persistent and caused widespread disease. Some viruses tended to be geographically restricted to a given area (CAV in California and MX97-8147 in Mexico), whereas others were widespread (Ark-DPI, Conn, DE072, and Mass). The Florida, Gray, Holte, Iowa, and JMK types were not detected during the 11-yr period, and no foreign virus types were detected in the United States. These data show that IBV variant viruses are consistently circulating in commercial poultry and are capable of causing disease outbreaks. Our observations highlight the importance of constantly monitoring IBV as well as other coronaviruses like severe acute respiratory syndrome-coronavirus that have the ability to change and emerge to cause disease in a susceptible host.

Emergence of a group 3 coronavirus through recombination

Abstract Analyses of turkey coronavirus (TCoV), an enteric disease virus that is highly similar to infectious bronchitis virus (IBV) an upper-respiratory tract disease virus in chickens, were conducted to determine the adaptive potential, and genetic changes associated with emergence of this group 3 coronavirus. Strains of TCoV that were pathogenic in poults and nonpathogenic in chickens did not adapt to cause disease in chickens. Comparative genomics revealed two recombination sites that replaced the spike gene in IBV with an unidentified sequence likely from another coronavirus, resulting in cross-species transmission and a pathogenicity shift. Following emergence in turkeys, TCoV diverged to different serotypes through the accumulation of mutations within spike. This is the first evidence that recombination can directly lead to the emergence of new coronaviruses and new coronaviral diseases, emphasizing the importance of limiting exposure to reservoirs of coronaviruses that can serve as a source of genetic material for emerging viruses.

Protection of chickens from infectious bronchitis by in ovo and intramuscular vaccination with a DNA vaccine expressing the S1 glycoprotein.

Abstract We have constructed a DNA vaccine (pDKArkS1-DPI) expressing the S1 glycoprotein (Arkansas DPI) of infectious bronchitis virus (IBV) to examine protective immunity after in ovo and intramuscular DNA immunization. Birds receiving in ovo DNA followed by live virus vaccination at 2 wk of age were 100% protected from clinical disease. Birds receiving only live virus vaccine or only in ovo DNA vaccination were ≤80% protected. IBV was detected up to 10 days postchallenge in unvaccinated control groups, whereas birds receiving in ovo DNA and live virus vaccination cleared IBV from tracheal samples before day 5 postchallenge. Transcription of the S1 gene was confirmed in lung tissue after in ovo vaccination by an antisense riboprobe, and the S1 protein was detected by immunohistology in the heart and bursa. In a separate experiment, birds were injected intramuscularly with either 50, 100, or 150 µg of the DNA vaccine at 1 day of age and then again with either 100, 200, or 300 µg of the DNA vaccine, respectively, at 14 days of age. At 10 days postchallenge, no clinical signs were observed and no challenge virus was reisolated from the birds vaccinated with 150 µg and 300 µg of DNA. Between DNA-vaccinated birds and nonvaccinated control birds, no statistical differences were observed for IBV-specific serum antibodies as detected by enzyme-linked immunosorbent assay or the virus neutralization test. These data indicate that DNA vaccination with the S1 gene either in ovo or intramuscularly can provide birds with some protection against clinical disease after homologous IBV challenge.

Typing of field isolates of infectious bronchitis virus based on the sequence of the hypervariable region in the S1 gene

A universal primer set was developed that amplifies a region covering hypervariable region (HVR) 1 and HVR 2 in the S1 gene of the infectious bronchitis virus (IBV). The universality of this primer set was confirmed by testing the reference strains of different serotypes or variants of the IBV present in the United States. An approximately 450-bp region containing HVR 1 and HVR 2 of 7 untyped field isolates obtained in 1999 and 2000 was amplified. Direct sequencing followed by phylogenetic analysis on that region allowed us to type those field isolates that were not typable by reverse transcriptase–polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP). Furthermore, it was found that typing by phylogenetic analysis of that region correlates with virus neutralization results. Together with RT-PCR and RFLP, this method will serve as a fast typing method for IBV diagnosis.

Infectious Bronchitis Virus Detection in Allantoic Fluid using the Polymerase Chain Reaction and a DNA Probe

A rapid extraction procedure was developed to purify infectious bronchitis virus (IBV) RNA from the allantoic fluid of inoculated embryonating eggs. Reverse transcription of viral RNA and the polymerase chain reaction (PCR) were used to amplify the viral genome from eight different strains of IBV comprising five different serotypes. A biotinylated DNA probe, prepared to a sequence within the PCR amplification product of the Beaudette strain of IBV, was used in a dot-hybridization assay; it detected the amplification products of all of the IBV strains examined. Reverse transcription and PCR amplification were judged to be specific for IBV. This was because amplification products were not detected by agarose gel electrophoresis or by dot-hybridization when template used in the PCR was extracted from allantoic fluid and the chorioallantoic membrane of uninoculated embryonating eggs or from allantoic fluid of embryonating eggs inoculated with other chicken upper respiratory viruses.