Korean Journal of Veterinary Service (한국동물위생학회지)

The Korean Society of Veterinary Service (한국동물위생학회)
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Prevalence and co-infection status of three pathogenic porcine circoviruses (PCV2, PCV3, and PCV4) by a newly established triplex real-time polymerase chain reaction assay

  • Kim, Hye-Ryung (College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University) ;
  • Park, Jonghyun (College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University) ;
  • Kim, Won-Il (College of Veterinary Medicine, Jeonbuk National University) ;
  • Lyoo, Young S. (College of Veterinary Medicine, Konkuk University) ;
  • Park, Choi-Kyu (College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University)
  • Received : 2022.06.07
  • Accepted : 2022.06.12
  • Published : 2022.06.30
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Abstract

A novel porcine circovirus 4 (PCV4) was recently emerged in Chinese and Korean pig herds, which provided epidemiological situation where three pathogenic PCVs, PCV2, PCV3, and newly emerged PCV4, could co-infect pig herds in these countries. In this study, a new triplex quantitative real-time polymerase chain reaction (tqPCR) method was developed for the rapid and differential detection of these viruses. The assay specifically amplified each viral capsid gene, whereas no other porcine pathogenic genes were detected. The detection limit of the assay was below 10 copies/µL and the assay showed high repeatability and reproducibility. In the clinical evaluation using 1476 clinical samples from 198 Korean pig farms, the detection rates of PCV2, PCV3 and PCV4 by the tqPCR assay were 13.8%, 25.4%, and 3.8%, respectively, which were 100% agreement with those of previously reported monoplex qPCR assays for PCV2, PCV3, and PCV4, with a κ value (95% CI) of 1 (1.00~1.00). The prevalence of PCV2, PCV3, and PCV4 at the farm levels were 46.5%, 63.6%, and 19.7%, respectively. The co-infection analysis for tested pig farms showed that single infection rates for PCV2, PCV3, and PCV4 were 28.8%, 44.4%, and 9.6%, respectively, the dual infection rates of PCV2 and PCV3, PCV2 and PCV4, and PCV3 and PCV4 were 12.6%, 3.5%, and 5.1%, respectively, and the triple infection rate for PCV2, PCV3, and PCV4 was 1.5%. These results demonstrate that three pathogenic PCVs are widely spread, and their co-infections are common in Korean pig herds, and the newly developed tqPCR assay will be useful for etiological and epidemiological studies of these pathogenic PCVs.

Keywords

Acknowledgement

This work was supported by the Commercialization Promotion Agency for R&D Outcomes (COMPA) grant funded by the Korean Government (Ministry of Science and ICT) (R&D project No. 1711139487), the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through the Animal Disease Management Technology Development Program (321015-01-1-CG000), and "Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01561102)" funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA), Rural Development Administration (RDA), Republic of Korea.

References

  1. Broeders S, Huber I, Grohmann L, Berben G, Taverniers I, Mazzara M, Roosens N, Morisset D. 2014. Guidelines for validation of qualitative real-time PCR methods. Trends Food Sci Technol 37(2): 115-126. https://doi.org/10.1016/j.tifs.2014.03.008
  2. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4): 611-622. https://doi.org/10.1373/clinchem.2008.112797
  3. Chen N, Xiao Y, Li X, Li S, Xie N, Yan X, Li X, Zhu J. 2021. Development and application of a quadruplex real-time PCR assay for differential detection of porcine circoviruses (PCV1 to PCV4) in Jiangsu province of China from 2016 to 2020. Transbound Emerg Dis 68(3): 1615-1624. https://doi.org/10.1111/tbed.13833
  4. Chen W, Jiang D, Xiao L, Zhang P, Luo Y, Yang Z, Yao X, Wang Y, Wu X. 2022. Development of a real-time TaqMan PCR assay for the detection of porcine circovirus 4. J Vet Res 66(1): 29-33. https://doi.org/10.2478/jvetres-2022-0004
  5. Fux R, Sockler C, Link EK, Renken C, Krejci R, Sutter G, Ritzmann M, Eddicks M. 2018. Full genome characterization of porcine circovirus type 3 isolates reveals the existence of two distinct groups of virus strains. Virol J 15(1): 25. https://doi.org/10.1186/s12985-018-0929-3
  6. Hou CY, Xu T, Zhang LH, Cui JT, Zhang,YH, Li XS, Zheng LL, Chen HY. 2021. Simultaneous detection and differentiation of porcine circovirus 3 and 4 using a SYBR Green I-based duplex quantitative PCR assay. J Virol Methods 293: 114152. https://doi.org/10.1016/j.jviromet.2021.114152
  7. Kalendar R, Lee D, Schulman AH. 2011. Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis. Genomics 98(2): 137-144. https://doi.org/10.1016/j.ygeno.2011.04.009
  8. Kim DY, Kim HR, Park JH, Kwon NY, Kim JM, Kim JK, Park JH, Lee KK, Kim SH, Kim WI, Lyoo YS, Park CK. 2022a Detection of a novel porcine circovirus 4 in Korean pig herds using a loop-mediated isothermal amplification assay. J Virol Methods 299: 114350. https://doi.org/10.1016/j.jviromet.2021.114350
  9. Kim HR, Lim DR, Chae HG, Park JY, Kim SH, Lee KK, Lee C, Lyoo YS, Park CK. 2020. Advanced targetspecific probe-based real-time loop-mediated isothermal amplification assay for the rapid and specific detection of porcine circovirus 3. Transbound Emerg Dis 67(6): 336-2344.
  10. Kim HR, Park J, Park JH, Kim JM, Baek KS, Kim DY, Lyoo YS, Park CK. 2022b Development of a real-time polymerase chain reaction assay for reliable detection of a novel porcine circovirus 4 with an endogenous internal positive control. Korean J Vet Serv 45(1): 1-11. https://doi.org/10.7853/kjvs.2022.45.1.1
  11. Kim HR, Park YR, Lim DR, Park MJ, Park JY, Kim SH, Lee KK, Lyoo YS, Park CK. 2017. Multiplex real-time polymerase chain reaction for the differential detection of porcine circovirus 2 and 3. J Virol Methods 250: 11-16. https://doi.org/10.1016/j.jviromet.2017.09.021
  12. Klaumann F, Correa-Fiz F, Sibila M, Nunez JI, Segales J. 2019. Infection dynamics of porcine circovirus type 3 in longitudinally sampled pigs from four Spanish farms. Vet Rec 184(20): 619-619. https://doi.org/10.1136/vr.105219
  13. Ku X, Chen F, Li P, Wang Y, Yu X, Fan S, Qian P, Wu M, He Q. 2017. Identification and genetic characterization of porcine circovirus type 3 in China. Transbound Emerg Dis 64(3): 703-708. https://doi.org/10.1111/tbed.12638
  14. Kwiecien R, Kopp-Schneider A, Blettner M. 2011. Concordance analysis: part 16 of a series on evaluation of scientific publications. Dtsch Arztebl Int 108(30): 515-521.
  15. Kwon T, Yoo SJ, Park CK, Lyoo YS. 2017. Prevalence of novel porcine circovirus 3 in Korean pig populations. Vet Microbiol 207: 178-180. https://doi.org/10.1016/j.vetmic.2017.06.013
  16. Li X, Qiao M, Sun M, Tian K. 2018. A duplex real-time PCR assay for the simultaneous detection of porcine circovirus 2 and circovirus 3. Virol Sin 33(2): 181-186. https://doi.org/10.1007/s12250-018-0025-2
  17. Navarro E, Serrano-Heras G, Castano MJ, Solera J. 2015. Real-time PCR detection chemistry. Clin Chim Acta 439: 231-250. https://doi.org/10.1016/j.cca.2014.10.017
  18. Nguyen VG, Do HQ, Huynh TML, Park YH, Park BK, Chung HC. 2021. Molecular-based detection, genetic characterization and phylogenetic analysis of porcine circovirus 4 from Korean domestic swine farms. Transbound Emerg Dis 69(2): 538-548.
  19. Opriessnig T, Meng XJ, Halbur PG. 2007. Porcine circovirus type 2-associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies. J Vet Diagn Invest 19(6): 591-615. https://doi.org/10.1177/104063870701900601
  20. Palinski R, Pineyro P, Shang P, Yuan F, Guo R, Fang Y, Byers E, Hause BM. 2017. A novel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure. J Virol 91(1): e01879-16.
  21. Park CK, Lee KK, Kim HS. 2004. Genetic characterization of porcine circovirus 2 Korean isolates. Korean J Vet Res 44: 571-579.
  22. Park YR, Kim HR, Kim SH, Lee KK, Lyoo YS, Yeo SG, Park CK. 2018. Loop-mediated isothermal amplification assay for the rapid and visual detection of novel porcine circovirus 3. J Virol Methods 253: 26-30. https://doi.org/10.1016/j.jviromet.2017.12.006
  23. Phan TG, Giannitti F, Rossow S, Marthaler D, Knutson TP, Li L, Deng X, Resende T, Vannucci F, Delwart E. 2016. Detection of a novel circovirus PCV3 in pigs with cardiac and multi-systemic inflammation. Virol J 13(1): 184. https://doi.org/10.1186/s12985-016-0642-z
  24. Rodriguez A, Rodriguez M, Cordoba JJ, Andrade MJ. 2015. Design of primers and probes for quantitative real-time PCR methods. Methods Mol Biol 1275: 31-56. https://doi.org/10.1007/978-1-4939-2365-6_3
  25. Rosario K, Breitbart M, Harrach B, Segales J, Delwart E, Biagini P, Varsani A. 2017. Revisiting the taxonomy of the family Circoviridae: establishment of the genus Cyclovirus and removal of the genus Gyrovirus. Arch Virol 162(5): 1447-1463. https://doi.org/10.1007/s00705-017-3247-y
  26. Saraiva GL, Vidigal PMP, Assao VS, Fajardo MLM, Loreto ANS, Fietto JLR, Bressan GC, Lobato ZIP, Almeida MR, Silva-Junior A. 2019. Retrospective detection and genetic characterization of porcine circovirus 3 (PCV3) strains identified between 2006 and 2007 in Brazil. Viruses 11(3): 201. https://doi.org/10.3390/v11030201
  27. Segales J. 2012. Porcine circovirus type 2 (PCV2) infections: clinical signs, pathology and laboratory diagnosis. Virus Res 164(1-2): 10-19. https://doi.org/10.1016/j.virusres.2011.10.007
  28. Stadejek T, Wozniak A, Milek D, Biernacka K. 2017. First detection of porcine circovirus type 3 on commercial pig farms in Poland. Transbound Emerg Dis 64(5): 1350-1353. https://doi.org/10.1111/tbed.12672
  29. Sun W, Du Q, Han Z, Bi J, Lan T, Wang W, Zheng M. 2021. Detection and genetic characterization of porcine circovirus 4 (PCV4) in Guangxi, China. Gene 773: 145384. https://doi.org/10.1016/j.gene.2020.145384
  30. Tian RB, Zhao Y, Cui JT, Zheng HH, Xu T, Hou CY, Wang ZY, Li XS, Zheng LL, Chen HY. 2021. Molecular detection and phylogenetic analysis of porcine circovirus 4 in Henan and Shanxi provinces of China. Transbound Emerg Dis 68(2): 276-282. https://doi.org/10.1111/tbed.13714
  31. Tischer I, Gelderblom H, Vettermann W, Koch MA. 1982. A very small porcine virus with circular single-stranded DNA. Nature 295(5844): 64-66. https://doi.org/10.1038/295064a0
  32. Tochetto C, Lima DA, Varela APM, Loiko MR, Paim WP, Scheffer CM, Herpich JI, Cerva C, Schmitd C, Cibulski SP, Santos AC, Mayer FQ, Roehe PM. 2018. Full-Genome sequence of porcine circovirus type 3 recovered from the serum of sows with stillbirths in Brazil. Transbound Emerg Dis 65(1): 5-9. https://doi.org/10.1111/tbed.12735
  33. Wang Y, Feng Y, Zheng W, Noll L, Porter E, Potter M, Cino G, Peddireddi L, Liu X, Anderson G, Bai J. 2019. A multiplex real-time PCR assay for the detection and differentiation of the newly emerged porcine circovirus type 3 and continuously evolving type 2 strains in the United States. J Virol Methods 269: 7-12. https://doi.org/10.1016/j.jviromet.2019.03.011
  34. Yang K, Jiao Z, Zhou D, Guo R, Duan Z, Tian Y. 2019. Development of a multiplex PCR to detect and discriminate porcine circoviruses in clinical specimens. BMC Infect Dis 19(1): 1-10. https://doi.org/10.1186/s12879-018-3567-x
  35. Zhang D, Bai C, Ge K, Li Y, Gao W, Jiang S, Wang Y. 2020a. Establishment of an SYBR Green-based real-time PCR assay for porcine circovirus type 4 detection. J Virol Methods, 285: 113963. https://doi.org/10.1016/j.jviromet.2020.113963
  36. Zhang HH, Hu WQ, Li JY, Liu TN, Zhou JY, Opriessnig T, Xiao CT. 2020b. Novel circovirus species identified in farmed pigs designated as porcine circovirus 4, Hunan province, China. Transbound Emerg Dis 67(3): 1057-1061. https://doi.org/10.1111/tbed.13446