Study on the biology of circovirus encoded proteins with special emphasis on pigeon circovirus

Pigeon circovirus (PiCV) is an important viral pathogen central to the development of young pigeon disease syndrome (YPDS). In this study, the Escherichia coli expression system was used to express the full-length native capsid protein (Cap) of PiCV, which was self-assembled into VLPs with a spherical morphology and diameters of 17–20 nm. The Cap was synthesised using a two‐step purification process resulting in high concentration and high purity than previously known. Polyclonal antibody against the Cap protein were induced after sheep immunised intramuscularly with VLPs combined with adjuvant. Polyclonal anti-PiCV Cap antibody was then used for the development of immunohistochemistry (IHC) techniques to detect viral antigen in paraffin-embedded, formalin-fixed tissue samples. These findings provide a better understanding for the recombinant expression and purification of the Cap protein of PiCV which has potential applications in immunodiagnostics, vaccine production and Cryo-EM reconstruction. A TaqMan based quantitative polymerase chain reaction (qPCR) targeting the conserved replication associated protein encoded gene (rep) was developed and validated for the detection of highly diverse PiCV in clinical samples. Ten-fold serial dilutions of pMCSG21 plasmid containing PiCV genomic insert were used to generate a standard curve with quantities of PiCV DNA molecules ranging from 2.5 × 10⁹ to 1 DNA molecules/μL. The limit of detection of the new TaqMan qPCR was lower (2 plasmid copies) than that for a previously published SYBR green based PiCV qPCR (5 plasmid copies). Assessments of 100% specificity and 100% sensitivity were attained based on the qPCR results with panel of 60 samples (known PiCV positive, n=30; known PiCV negative, n=20; samples positive to Beak and feather disease virus (BFDV), n=5 and samples positive to Canine circovirus, n=5). Co-efficient of variation (CV) for Ct value ranged between 0.27% to 0.78% in the intra-assay and 1.84% to 2.87% in inter-assays. The detection of PiCV was enhanced by 16% over that obtained by established conventional PCR. Testing of clinical samples from pigeons showed that PiCV loads were high in the liver (<2.38 × 10¹¹ copies/μL), oropharynx (<2.84 × 10⁹ copies/μL) and bursa of Fabricius (<2.10 × 10⁹ copies/μL). Cloacal swabs samples also yielded high viral loads in clinically affected birds (8.98 × 10⁸ copies/μL). TaqMan qPCR demonstrated higher sensitivity for detection of highly diverse PiCV compared to the existing conventional PCR method. Like other circoviruses PiCV has been considered to be genetically diverse and prone to recombination. Current paleoviral evidence suggest towards a probable Gondwanan origin of avian circoviruses, paralleling the evolution and distribution of their hosts. In this study a phylogenetic analysis of new PiCV sequences (n=15) from a diverse group of extant and aberrant hosts revealed no biogeographic or host adapted structure of PiCV in the Australian avifauna. Investigation of spillover infection of PiCV in plumed whistling duck, blue billed duck and Australian magpie uncovered the host‐generalist and host‐switching characteristics of PiCV. In addition, analysis of host-virus phylogeny demonstrated patterns of avian circovirus spillover across different Orders of birds. Furthermore, a naturally occurring CoHV-1 and PiCV coinfection in a flock of racing pigeons (Columba livia) was also investigated using quantitative polymerase chain reaction (qPCR). Clinically affected pigeons had suppurative stomatitis, pharyngitis, vesicular dermatitis, cloacitis, otitis externa, tympanitis, otitis media and diffuse granulomatous meningitis with eosinophilic intranuclear inclusions consistent with herpesvirus infection later confirmed by quantitative PCR and DNA sequencing. Large numbers of amphophilic, botryoid intracytoplasmic inclusions were present in the skin, oral mucosa and bursa of Fabricius confirmed as PiCV infection by immunohistochemistry. Concurrent infection was widespread in visceral organs of the affected birds with highest concentration detected in oropharynx and in liver samples for CoHV-1 and PiCV respectively. Interestingly, pigeon with only CoHV-1 was not detected regardless of clinical status. These results suggests that both viral copy numbers were significantly higher (p < 0.0001) in clinically affected pigeons compared to asymptomatic qPCR positive birds and CoHV-1 induced pathology might have been aggravated by concurrent PiCV infection. Since no structural information is currently available for any avian circovirus replication associated protein (Rep), an E. coli based recombinant expression and purification method for endonuclease domain and full-length native BFDV Rep was developed and optimised for downstream structural biology applications. Each protein was synthesised using a two‐step purification process resulting in high concentration and high purity. For the first time, high-resolution structure of BFDV Rep protein was resolved through cryo-EM reconstruction at 3.4 Å. The structure demonstrates full-length Rep assembles in a hexameric ring possess oligonucleotide on the helicase domain and allowed the identification of three residues implicated in this interaction. The implication of these residues has been tested by the expression and purification of 3 (three) point mutants to further probe its capacity for ssDNA binding and replication. EMSA assay showed full-length native and mutant Rep protein possesses affinity to ssDNA. Furthermore, significant nicking and unwinding activity was also observed in Rep K252D. This study significantly enhances the knowledge of avian circovirus replication as full-length native BFDV Rep structure has been constructed for the first time using high resolution cryo-EM technique.