Application of high-resolution melt curve analysis for classification of infectious bronchitis viruses in field specimens.
OBJECTIVE
A real-time polymerase chain reaction (PCR)/high-resolution melt (HRM) curve analysis protocol was developed in our laboratory to differentiate infectious bronchitis (IB) virus reference strains. In the current study, this method was used to detect and classify IB viruses in field submissions.


METHODS
Over an 11-month period samples from 40 cases of suspected IB virus were received and 17 submissions were positive for IB virus by polymerase chain reaction. HRM curve analysis classified each strain as subgroup 1, 2 or 3 strain (12 submissions) or a strain that was unable to be classified (5 submissions). The 3' untranslated region (UTR) and partial S1 gene nucleotide sequences for the 17 IB virus strains were determined and their identity with those of the relative reference strains compared to confirm the classifications generated using the HRM curve analysis.


RESULTS
Of the 12 IB field viruses classified as subgroup 1, 2, or 3 using HRM curve analysis, the 3'UTR and S1 gene nucleotide sequences had identities ≥99% with the respective subgroup reference strain. Analysis of the 3' UTR and S1 gene nucleotide sequences for the five IB virus strains that could not be classified indicated that four belonged to one of the subgroups, and one was a potential recombinant strain (between strains from subgroups 2 and 3). A novel recombinant strain was also detected.


CONCLUSIONS
HRM curve analysis can rapidly assign the majority of IB viruses present in field submissions to known subgroups. Importantly, HRM curve analysis also identified variant genotypes that require further investigation.
Hewson
K A
KA
Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia. khewson@unimelb.edu.au
Browning
G F
GF
Devlin
J M
JM
Ignjatovic
J
J
Noormohammadi
A H
AH
D020413:3' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D002645:Chickens / Q000821:virology*; D018352:Coronavirus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D004279:DNA, Viral; D005784:Gene Amplification; D005838:Genotype; D001351:Infectious bronchitis virus / Q000145:classification* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010802:Phylogeny; D011201:Poultry Diseases / Q000175:diagnosis / Q000821:virology*; D012367:RNA, Viral / Q000235:genetics
D020413:3' Untranslated Regions; D004279:DNA, Viral; D012367:RNA, Viral