Cloning and expression of a truncated pigeon circovirus capsid protein suitable for antibody detection in infected pigeons

Abstract

Infections with pigeon circovirus (PiCV) (also termed columbid circovirus) occur in meat and racing pigeons (Columba livia) of all ages and have been reported worldwide. A PiCV infection is associated with immunosuppression and the development of young pigeon disease syndrome. An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of virus-specific serum antibody was developed for research purposes. In the absence of a method to propagate PiCV in cell culture, the assay was based on a recombinant truncated capsid protein (rCapPiCV) produced by overexpression in Escherichia coli. A 6xHis-Tag was fused to the N-terminus of the protein to facilitate purification by metal affinity chromatography and detection by anti-His antibody. PiCV-negative and PiCV-positive control sera were generated by inoculation of pigeons with tissue homogenate containing PiCV, followed by five weekly blood sample collections. Western blotting of the immune serum revealed a specific protein band of approximately 32 kDa, which was absent in the pre-immune sera. Using rCapPiCV as antigen in an indirect ELISA, PiCV-specific antibody was detected in sera of the experimentally PiCV-infected pigeons collected at 1 to 5 weeks post infection. By testing 118 field sera collected in the years 1989, 1991, 1994 and 2008 in the rCapPiCV ELISA, virus-specific antibody was detected in 89 (75%) of the sera. The results obtained demonstrate that the rCapPiCV-based indirect ELISA is able to detect PiCV-specific antibodies in pigeon sera and may be a useful tool for PiCV serodiagnosis.

Occurrence and genetic diversity of pigeon circovirus strains in Poland

“…So far, it is known that in circovirus infection of the pigeon organism, the arising antibodies are directed against the capsid protein (Daum et al, 2009;Duchatel et al, 2011). Based on previous studies the authors of this publication agree with certain hypotheses, which argue that changes in capsid protein structure may have an influence on the formation of resistance against this virus (Cságola et al, 2012).…”

The epidemiology, molecular characterization and clinical pathology of circovirus infections in pigeons – current knowledge

“…A specific protocol for the prevention of PiCV infections in pigeons has not yet been developed due to the absence of vaccines targeting this pathogen. In recent years, attempts have been made to develop a recombinant PiCV capsid protein for diagnostic tests and a subunit vaccine against the PiCV (Daum et al 2009;Lai et al 2014;Stenzel et al 2016). This vaccine could protect pigeons against PiCV infection and, possibly, lower the prevalence of YPDS.…”

“…Daum et al (2009) conducted a retrospective study of pigeon sera obtained in 1989, 1991 and 2008 and found anti-PiCV antibodies in 78% (61%-90%) of the tested samples on average. A recent serological study of Polish domestic pigeons from selected breeding flocks revealed the presence of anti-PiCV antibodies in 72% birds on average, regardless of their infection status (average prevalence of 36%) (Stenzel et al 2016).…”

Recurrent positive selection and heterogeneous codon usage bias events leading to coexistence of divergent pigeon circoviruses

“…The capsid protein of PiCV recognizes PiCV-specific antibodies in pigeon sera and can be used in the serodiagnosis of PiCV (Daum et al, 2009). Thus, the capsid protein mediates the induction of immune responses and has often been used to evaluate genetic changes between PiCV isolates (Stewart et al, 2006;Todd et al, 2008).…”