Development of real-time multiplex PCR analysis for detection of infectious bursal disease virus and infectious anemia virus in chickens

Infectious bursal disease and infectious anemia of chickens are significant and pressing problems in the modern poultry industry. The infectious bursal disease virus (IBDV) and chicken infectious anemia virus (CIAV) are highly prevalent, causing severe damage to lymphoid organs and tissues. This results in significant immunosuppression, increasing poultry susceptibility to secondary infections, reducing production performance, diminishing the efficacy of preventive measures, and raising economic losses. Currently, these diseases primarily occur in subclinical forms, against a vaccine-induced immunity background, and in co-infections. They are often accompanied by secondary infections and lack distinctive clinical signs, complicating diagnosis. We developed a multiplex real-time PCR assay for simultaneous detection of both viruses in a single sample. The assay employs primers targeting highly conserved overlapping regions of the IBDV and CIAV genomes, with TaqMan probes for detection. Analytical sensitivity testing using 10-fold serial dilutions confirmed high coefficients of determination and amplification efficiency. The assay demonstrates high specificity, with no cross-reactivity or nonspecific amplification. Its performance metrics (determination coefficients, reaction efficiency, and low intra- and inter-assay variability) are comparable to single-assays for IBDV and CIAV. The multiplex format significantly reduces processing time, minimizes contamination risks, and maintains high sensitivity and specificity. This PCR assay is expected to enable rapid and accurate identification of IBDV and CIAV, greatly improving diagnostic efficiency.

1. J. Hoerr. Clinical aspects of immunosuppression in poultry. Avian Dis. 54 (2010): 2–15. URL: https://doi.org/10.1637/8909-043009-Review.1.

2. Gimeno IM, Schat KA. Virus-Induced Immunosuppression in Chickens. Avian Dis. 2018 Sep;62(3):272-285. doi:10.1637/11841-041318-Review.1. URL: https://pubmed.ncbi.nlm.nih.gov/30339511/.

3. M. E. Dmitrieva, A. S. Dubovoi, V. A.Manuvera [et al.]. Biological properties of infectious bursal disease virus. Research Journal of Pharmaceutical, Biological and Chemical Sciences. 2018. Vol. 9, No. 6: 1891-1896. Access mode: https://elibrary.ru/item.asp?id=38674853.

4. Shirokov D. A., Manuvera V. A., Lazarev V. N. [et al.]. Complete genome sequence of a novel very virulent strain of infectious bursal disease virus circulating in Russia. Microbiology Resource Announcements. 2018. Vol. 7, No.20: e01084-18. Access mode: https://elibrary.ru/item.asp?id=38618595.

5. Shirokov D. A., Manuvera V. A., Lazarev V. N. [et al.]. Generation of recombinant VP3 protein of infectious bursal disease virus in three different expression systems, antigenic analysis of the obtained polypeptides and development of an ELISA test. Archives of Virology. 2020. Vol. 165, No. 7: 1611-1620. - DOI 10.1128/MRA.01084-18. Access mode: https://elibrary.ru/item.asp?id=43274990.

6. Lin W, Zhang Z, Xu Z. et al. The association of receptor of activated protein kinase C 1 (RACK1) with infectious bursal disease virus viral protein VP5 and voltagedependent anion channel 2 (VDAC2) inhibits apoptosis and enhances viral replication. J Biol Chem. 2015. Mar 27; 290 (13): 8500-10. URL: https://www.sciencedirect.com/science/article/pii/S0021925820639904?via%3Dihub.

7. Rosario, M. Breitbart, B. Harrach, J. Segalés, E. Delwart, P. Biagini, A. Varsani. Revisiting the taxonomy of the family Circoviridae: establishment of the genus Cyclovirus and removal of the genus Gyrovirus. Arch. Virol. 162 (2017): 1447–1463. URL: https://link.springer.com/article/10.1007/s00705-017-3247-y.

8. Claudia Techera, Ana Marandino, Gonzalo Tomás, Sofía Grecco, Martín Hernández, Diego Hernández, Yanina Panzera & Ruben Pérez. Origin, spreading and genetic variability of chicken anemia virus. Avian Pathology. 2021. T.50:4: 311-320. URL: https://www.tandfonline.com/doi/full/10.1080/03079457.2021.1919289.

9. Lai GH, Lin MK, Lien YY, Cheng JH, Sun FC, Lee MS, Chen HJ, Lee MS. Characterization of the DNA binding activity of structural protein VP1 from chicken anemia virus. BMC Vet Res. 2018. May 4. - T. 14 (1): 155. URL: https://bmcvetres.biomedcentral.com/articles/10.1186/s12917-018-1465-5.

10. E. Eregae, C. E. Dewey, S. A. McEwen, R. Ouckama, D. Ojkić, M. T. Guerin. Flock prevalence of exposure to avian adenoassociated virus, chicken anemia virus, fowl adenovirus, and infectious bursal disease virus among Ontario broiler chicken flocks. Avian Dis. 2014 (58): 71–77. URL: https://pubmed.ncbi.nlm.nih.gov/24758116/.

11. Abgaryan, S. R. Molecular biological methods for diagnosing bird diseases / S. R. Abgaryan // Current issues of veterinary medicine and laboratory diagnostics: Proceedings of the international scientific and practical conference dedicated to the 100th anniversary of the birth of Professor V.V. Rudakov, St. Petersburg, May 25–26, 2023. – St. Petersburg: St. Petersburg State University of Veterinary Medicine, 2023. – Pp. 3–5. – Access mode: https://elibrary.ru/item.asp?id=54023930.

12. Marandino, G. Tomás, M. Hernández, Y. Panzera, M.I. Craig, A. Vagnozzi, F. Vera, C. Techera, S. Grecco, A. Banda, D. Hernández, R. Pérez. Development of RT-qPCR assays for the specific identification of two major genotypes of avian infectious bronchitis virus. J. Virol. Methods. 2016. 235: 21–25. URL: https://www.sciencedirect.com/science/article/pii/S0166093416300325?via%3Dihub.