Kylie A. Hewson

Rapid detection and non-subjective characterisation of infectious bronchitis virus isolates using high-resolution melt curve analysis and a mathematical model

Infectious bronchitis virus (IBV) is a coronavirus that causes upper respiratory, renal and/or reproductive diseases with high morbidity in poultry. Classification of IBV is important for implementation of vaccination strategies to control the disease in commercial poultry. Currently, the lengthy process of sequence analysis of the IBV S1 gene is considered the gold standard for IBV strain identification, with a high nucleotide identity (e.g. ≥95%) indicating related strains. However, this gene has a high propensity to mutate and/or undergo recombination, and alone it may not be reliable for strain identification. A real-time polymerase chain reaction (RT-PCR) combined with high-resolution melt (HRM) curve analysis was developed based on the 3′UTR of IBV for rapid detection and classification of IBV from commercial poultry. HRM curves generated from 230 to 435-bp PCR products of several IBV strains were subjected to further analysis using a mathematical model also developed during this study. It was shown that a combination of HRM curve analysis and the mathematical model could reliably group 189 out of 190 comparisons of pairs of IBV strains in accordance with their 3′UTR and S1 gene identities. The newly developed RT-PCR/HRM curve analysis model could detect and rapidly identify novel and vaccine-related IBV strains, as confirmed by S1 gene and 3′UTR nucleotide sequences. This model is a rapid, reliable, accurate and non-subjective system for detection of IBVs in poultry flocks.

Detection of Avian Nephritis Virus in Australian Chicken Flocks

Abstract Avian nephritis virus (ANV) is thought to infect poultry flocks worldwide, but no confirmed case has been reported in Australia. The first such case is described in this study. Cases of young chickens with clinical signs of dehydration and diarrhea were submitted to our laboratory and histopathology detected interstitial nephritis. Vaccine strains of infectious bronchitis virus were detected in some of these cases but were not considered to be the causative agent. A total of seven fresh submissions from broiler chicken flocks were collected at 8–11 days of age. Degenerate PCR primers were designed based on published ANV polymerase gene sequences and used to analyze historic cases as well as the fresh submissions. Six of the seven fresh submissions, and one historic case, were positive for ANV with nucleotide sequencing confirming these results. These results establish ANV as an infectious pathogen circulating in Australian poultry.

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. Procedure  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. Conclusion  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.

Evaluation of a novel strain of infectious bronchitis virus emerged as a result of spike gene recombination between two highly diverged parent strains

The emergence of new variant strains of the poultry pathogen infectious bronchitis virus (IBV) is continually reported worldwide, owing to the labile nature of the large single-stranded RNA IBV genome. High resolution melt curve analysis previously detected a variant strain, N1/08, and the present study confirmed that this strain had emerged as a result of recombination between Australian subgroup 2 and 3 strains in the spike gene region, in a similar manner reported for turkey coronaviruses. The S1 gene for N1/08 had highest nucleotide similarity with subgroup 2 strains, which is interesting considering subgroup 2 strains have not been detected since the early 1990s. SimPlot analysis of the 7.2-kb 3′ end of the N1/08 genome with the same region for other Australian reference strains identified the sites of recombination as immediately upstream and downstream of the S1 gene. A pathogenicity study in 2-week-old chickens found that N1/08 had similar pathogenicity for chicken respiratory tissues to that reported for subgroup 2 strains rather than subgroup 3 strains. The results of this study demonstrate that recombination is a mechanism utilized for the emergence of new strains of IBV, with the ability to alter strain pathogenicity in a single generation.

Infectious bronchitis viruses with naturally occurring genomic rearrangement and gene deletion

Infectious bronchitis viruses (IBVs) are group III coronaviruses that infect poultry worldwide. Genetic variations, including whole-gene deletions, are key to IBV evolution. Australian subgroup 2 IBVs contain sequence insertions and multiple gene deletions that have resulted in a substantial genomic divergence from international IBVs. The genomic variations present in Australian IBVs were investigated and compared to those of another group III coronavirus, turkey coronavirus (TCoV). Open reading frames (ORFs) found throughout the genome of Australian IBVs were analogous in sequence and position to TCoV ORFs, except for ORF 4b, which appeared to be translocated to a different position in the subgroup 2 strains. Subgroup 2 strains were previously reported to lack genes 3a, 3b and 5a, with some also lacking 5b. Of these, however, genes 3b and 5b were found to be present but contained various mutations that may affect transcription. In this study, it was found that subgroup 2 IBVs have undergone a more substantial genomic rearrangements than previously thought.

The presence of viral subpopulations in an infectious bronchitis virus vaccine with differing pathogenicity--a preliminary study.

Abstract There are currently four commercially available vaccines in Australia to protect chickens against infectious bronchitis virus (IBV). Predominantly, IBV causes clinical signs associated with respiratory or kidney disease, which subsequently cause an increase in mortality rate. Three of the current vaccines belong to the same subgroup (subgroup 1), however, the VicS vaccine has been reported to cause an increased vaccinal reaction compared to the other subgroup 1 vaccines. Molecular anomalies detected in VicS suggested the presence of two major subspecies, VicS-v and VicS-del, present in the commercial preparation of VicS. The most notable anomaly is the absence of a 40 bp sequence in the 3′UTR of VicS-del. In this investigation, the two subspecies were isolated and shown to grow independently and to similar titres in embryonated chicken eggs. An in vivo investigation involved 5 groups of 20 chickens each and found that VicS-del grew to a significantly lesser extent in the chicken tissues collected than did VicS-v. The group inoculated with an even ratio of the isolated subspecies scored the most severe clinical signs, with the longest duration. These results indicate the potential for a cooperative, instead of an expected competitive, relationship between VicS-v and VicS-del to infect a host, which is reminiscent of RNA viral quasi-species.

Survey of captive parrot populations around Port Phillip Bay, Victoria, Australia, for psittacine beak and feather disease virus, avian polyomavirus and psittacine adenovirus.

OBJECTIVE This study investigated the prevalence of psittacine beak and feather disease virus (BFDV), avian polyomavirus (APV) and psittacine adenovirus (PsAdV) in captive psittacine birds around Port Phillip Bay, Victoria, Australia. METHODS Samples of fresh droppings were collected from 118 psittacine birds (109 clinically normal and 9 with feather abnormalities) from 11 avaries in different locations and were used for detection of BFDV, APV and PsAdV using PCR. RESULTS BFDV, APV and PsAdV were detected in 31%, 13% and 4%, respectively, of the specimens tested. One budgerigar was found to be co-infected with BFDV and PsAdV. At least one sample tested positive for BFDV at each location. CONCLUSION This is the first report of the prevalence of BFDV, APV and PsAdV in Victoria and provides a foundation for future studies examining the influence of these viruses on the health of aviary birds in Victoria.

Analysis of the complete genomic sequences of two virus subpopulations of the Australian infectious bronchitis virus vaccine VicS

Although sequencing of the 3′ end of the genome of Australian infectious bronchitis viruses (IBVs) has shown that their structural genes are distinct from those of IBVs found in other countries, their replicase genes have not been analysed. To examine this, the complete genomic sequences of the two subpopulations of the VicS vaccine, VicS-v and VicS-del, were determined. Compared with VicS-v, the more attenuated VicS-del strain had two non-synonymous changes in the non-structural protein 6 (nsp6), a transmembrane (TM) domain that may participate in autocatalytic release of the 3-chymotrypsin-like protease, a polymorphic difference at the end of the S2 gene, which coincided with the body transcription-regulating sequence (B-TRS) of mRNA 3 and a truncated open reading frame for a peptide encoded by gene 4 (4b). These genetic differences could be responsible for the differences between these variants in pathogenicity in vivo, and replication in vitro. Phylogenetic analysis of the whole genome showed that VicS-v and VicS-del did not cluster with strains from other countries, supporting the hypothesis that Australian IBV strains have been evolving independently for some time, and analyses of individual polymerase peptide and S glycoprotein genes suggested a distant common ancestor with no recent recombination. This study suggests the potential role of the TM domain in nsp6, the integrity of the S2 protein and the B-TRS 3, and the putative accessory protein 4b, as well as the 3′ untranslated region, in the virulence and replication of IBV and has provided a better understanding of relationships between the Australian vaccine strain of IBV and those used elsewhere.

Assessment of the potential relationship between egg quality and infectious bronchitis virus infection in Australian layer flocks

Objective This investigation aimed to determine if there was a relationship between the production of eggs with poor internal quality, as measured by poor Haugh units, by Australian layer flocks and the detection of infectious bronchitis virus (IBV) in the hens. Other risk factors including flock size, flock type, flock age, chicken breed and vaccination frequency were also assessed. Methods The study group comprised 17 flocks from 14 farms. Data relating to the factors investigated were requested on a regular basis. The Haugh unit data were used to grade eggs as good or poor based on the age and flock at the time of data collection. Cloacal swabs were collected from 20 chickens in each flock approximately every 6 weeks. Results IBV was detected from a majority of the flocks and in 68% of cases the IBV strain detected was an A‐vaccine‐related field strain. Three variant strains were detected. Detection of IBV in a flock, the farm type and flock size were identified as potential risk factors for the production of eggs with poor Haugh units. Conclusion IBV is prevalent in Australian layer flocks, but infection was primarily subclinical. The results complement previous reports indicating that there are many potential risk factors for the production of eggs with poor Haugh units.

High-resolution melt curve analysis to confirm the presence of co-circulating isolates of avian nephritis virus in commercial chicken flocks

Avian Nephritis Virus (ANV) has been implicated in poor growth and renal disease of young chickens. This paper describes the development of a reverse-transcriptase polymerase chain reaction for the detection of ANV in commercial meat chickens and the use of high-resolution melt curves to detect the presence of genetically different ANVs. Pooled cloacal swabs from both healthy and ill commercial chicken broiler flocks were tested for the presence of ANV using a combination of polymerase chain reaction, molecular cloning, high-resolution melt curve analysis and sequencing. Except for one, all specimens were found to contain two genetically different ANVs. Phylogenetic analysis of the capsid amino acid sequences revealed the presence of four of six groups of ANV identified previously in other countries as well as in two novel groups of ANV. Phylogenetic analysis of nucleotide sequences of partial polymerase, capsid and 3′ untranslated regions reveal that the genes of individual ANV virus isolates have different ancestors. This was shown to be due to a template-switching event in the capsid gene that resulted in the 3′ end of the capsid gene and the 3′ untranslated region of one ANV isolate being transferred to another ANV. These results reveal that infection of chicken flocks with multiple ANV isolates is common and this needs to be taken into consideration in diagnosis of ANV using molecular techniques and in future epidemiological investigations.