Porcine deltacoronavirus (PDCoV) and porcine sapelovirus (PSV) are two viruses that can cause diarrhoea in pigs and bring great economic loss to the pig industry. In this research, a duplex real-time quantitative polymerase chain reaction (qPCR) assay based on SYBR Green I was developed to simultaneously detect PDCoV and PSV. No specific melting peaks were found in other porcine diarrhoea-associated viruses, indicating that the method developed in this study had good specificity. The detection limits of PDCoV and PSV were 1.0 × 101 copies μl-1 and 1.0 × 102 copies μl-1, respectively. The duplex real-time qPCR assay tested two hundred and three (203) intestinal and faecal samples collected from diarrhoeal and asymptomatic pigs. The positive rates of PDCoV and PSV were 20.2% and 23.2%, respectively. The co-infection rate of PDCoV and PSV was 13.8%. To evaluate the accuracy of the developed method, conventional PCR and singular TaqMan real-time qPCR assays for PDCoV/PSV were also used to detect the samples. The results showed that the duplex real-time qPCR assay was consistent with the singular assays, but its sensitivity was higher than conventional PCR methods. This duplex real-time qPCR assay provides a rapid, sensitive and reliable method in a clinic to simultaneously detect PDCoV and PSV.

College of Veterinary Medicine Henan Agricultural University Zhengzhou Henan P R China

See more in PubMed

Arruda PH, Arruda BL, Schwartz KJ, Vannucci F, Resende T, Rovira A, Sundberg P, Nietfeld J, Hause BM. Detection of a novel sapelovirus in central nervous tissue of pigs with polioencephalomyelitis in the USA. Transbound Emerg Dis. 2017 Apr;64(2):311-5. PubMed

Boley PA, Alhamo MA, Lossie G, Yadav KK, Vasquez-Lee M, Saif LJ, Kenney SP. Porcine deltacoronavirus infection and transmission in poultry, United States. Emerg Infect Dis. 2020 Feb;26(2):255-65. PubMed PMC

Boros A, Laszlo Z, Pankovics P, Marosi A, Albert M, Csagola A, Biro H, Fahsbender E, Delwart E, Reuter G. High prevalence, genetic diversity and a potentially novel genotype of Sapelovirus A (Picornaviridae) in enteric and respiratory samples in Hungarian swine farms. J Gen Virol. 2020 Jun;101(6):609-21. PubMed PMC

Chen Q, Gauger P, Stafne M, Thomas J, Arruda P, Burrough E, Madson D, Brodie J, Magstadt D, Derscheid R, Welch M, Zhang J. Pathogenicity and pathogenesis of a United States porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets. Virology. 2015 Aug;482:51-9. PubMed PMC

Ding G, Fu Y, Li B, Chen J, Wang J, Yin B, Sha W, Liu G. Development of a multiplex RT-PCR for the detection of major diarrhoeal viruses in pig herds in China. Transbound Emerg Dis. 2020 Mar;67(2):678-85. PubMed PMC

Ding Q, Yan X, Zhang H, Li Q, Zhang Y, Shu X, Shan F, Li L, Hu H. [Establishment and preliminary clinical application of a multiple RT-PCR test for the detection of porcine deltacoronavirus, porcine epidemic diarrhea virus and porcine sapelovirus]. Chin J Prev Vet Med. 2021 Jun;43(6):615-20+632. Chinese.

Donin DG, de Arruda Leme R, Alfieri AF, Alberton GC, Alfieri AA. First report of porcine teschovirus (PTV), porcine sapelovirus (PSV) and enterovirus G (EV-G) in pig herds of Brazil. Trop Anim Health Prod. 2014 Mar;46(3):523-8. PubMed

Harima H, Kajihara M, Simulundu E, Bwalya E, Qiu Y, Isono M, Okuya K, Gonzalez G, Yamagishi J, Hang’ombe BM, Sawa H, Mweene AS, Takada A. Genetic and biological diversity of porcine sapeloviruses prevailing in Zambia. Viruses. 2020 Feb 5;12(2):180. PubMed PMC

Jiang C, He H, Zhang C, Zhang X, Han J, Zhang H, Luo Y, Wu Y, Wang Y, Ge B, Xu J. One-step triplex reverse-transcription PCR detection of porcine epidemic diarrhea virus, porcine sapelovirus, and porcine sapovirus. J Vet Diagn Invest. 2019 Nov;31(6):909-12. PubMed PMC

Kumari S, Ray PK, Singh R, Desingu PA, Sharma GT, Saikumar G. Development of a Taqman-based real-time PCR assay for detection of porcine sapelovirus infection in pigs. Anim Biotechnol. 2020 Jun;31(3):264-7. PubMed

Lamont PH, Betts AO. Studies on enteroviruses of the pig IV: Pathogenic swine orphan (ECSO) virus (V13) from the faeces of a pig. Res Vet Sci. 1960;1(2):152-9.

Le VP, Song S, An BH, Park GN, Pham NT, Le DQ, Nguyen VT, Vu TTH, Kim KS, Choe S, An DJ. A novel strain of porcine deltacoronavirus in Vietnam. Arch Virol. 2018 Jan;163(1):203-7. PubMed PMC

Lednicky JA, Tagliamonte MS, White SK, Elbadry MA, Alam MM, Stephenson CJ, Bonny TS, Loeb JC, Telisma T, Chavannes S, Ostrov DA, Mavian C, Beau De Rochars VM, Salemi M, Morris JG Jr. Independent infections of porcine deltacoronavirus among Haitian children. Nature. 2021 Dec;600(7887):133-7. PubMed PMC

Lee S, Lee C. Complete genome characterization of Korean porcine deltacoronavirus strain KOR/KNU14-04/2014. Genome Announc. 2014 Nov 26;2(6):e01191-14. PubMed PMC

Li W, Hulswit RJG, Kenney SP, Widjaja I, Jung K, Alhamo MA, van Dieren B, van Kuppeveld FJM, Saif LJ, Bosch BJ. Broad receptor engagement of an emerging global coronavirus may potentiate its diverse cross-species transmissibility. Proc Natl Acad Sci U S A. 2018 May 29;115(22):E5135-E43. PubMed PMC

Li B, Zheng L, Li H, Ding Q, Wang Y, Wei Z. Porcine deltacoronavirus causes diarrhea in various ages of field-infected pigs in China. Biosci Rep. 2019a Sep 16;39(9):BSR20190676. PubMed PMC

Li Y, Du L, Jin T, Cheng Y, Zhang X, Jiao S, Huang T, Zhang Y, Yan Y, Gu J, Zhou J. Characterization and epidemiological survey of porcine sapelovirus in China. Vet Microbiol. 2019b May;232:13-21. PubMed

Li Q, Zhao F, Ding Q, Zhang Y, Zheng L. [Establishment of a TaqMan fluorescent real-time quantitative PCR for detection for porcine sapelovirus]. J Northwest Sci Tech Univer Agric Forest. 2021 May;49(11):10-6. Chinese.

Liang Q, Zhang H, Li B, Ding Q, Wang Y, Gao W, Guo D, Wei Z, Hu H. Susceptibility of chickens to porcine deltacoronavirus infection. Viruses. 2019 Jun 21;11(6):573. PubMed PMC

Luo Y, Chen Y, Geng R, Li B, Chen J, Zhao K, Zheng XS, Zhang W, Zhou P, Yang XL, Shi ZL. Broad cell tropism of SADS-CoV in vitro implies its potential cross-species infection risk. Virol Sin. 2021 Jun;36(3):559-63. PubMed PMC

Ma L, Zeng F, Huang B, Cong F, Huang R, Ma J, Guo P. Development of a conventional RT-PCR assay for rapid detection of porcine deltacoronavirus with the same detection limit as a SYBR green-based real-time RT-PCR assay. Biomed Res Int. 2018 Nov 6;2018:5035139. PubMed PMC

Masuda T, Sunaga F, Naoi Y, Ito M, Takagi H, Katayama Y, Omatsu T, Oba M, Sakaguchi S, Furuya T, Yamasato H, Shirai J, Makino S, Mizutani T, Nagai M. Whole genome analysis of a novel picornavirus related to the enterovirus/sapelovirus supergroup from porcine feces in Japan. Virus Res. 2018 Sep 15;257:68-73. PubMed

Pan Z, Lu J, Wang N, He WT, Zhang L, Zhao W, Su S. Development of a TaqMan-probe-based multiplex real-time PCR for the simultaneous detection of emerging and reemerging swine coronaviruses. Virulence. 2020 Jan 1;11(1):707-18. PubMed PMC

Prodelalova J. The survey of porcine teschoviruses, sapeloviruses and enteroviruses B infecting domestic pigs and wild boars in the Czech Republic between 2005 and 2011. Infect Genet Evol. 2012 Oct;12(7):1447-51. PubMed

Son KY, Kim DS, Kwon J, Choi JS, Kang MI, Belsham GJ, Cho KO. Full-length genomic analysis of Korean porcine sapelovirus strains. PLoS One. 2014 Sep 17;9(9):e107860. PubMed PMC

Song D, Zhou X, Peng Q, Chen Y, Zhang F, Huang T, Zhang T, Li A, Huang D, Wu Q, He H, Tang Y. Newly emerged porcine deltacoronavirus associated with diarrhoea in swine in China: Identification, prevalence and full-length genome sequence analysis. Transbound Emerg Dis. 2015 Dec;62(6):575-80. PubMed PMC

Wang L, Byrum B, Zhang Y. Detection and genetic characterization of deltacoronavirus in pigs, Ohio, USA, 2014. Emerg Infect Dis. 2014 Jul;20(7):1227-30. PubMed PMC

Woo PC, Lau SK, Lam CS, Lau CC, Tsang AK, Lau JH, Bai R, Teng JL, Tsang CC, Wang M, Zheng BJ, Chan KH, Yuen KY. Discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus. J Virol. 2012 Apr;86(7):3995-4008. PubMed PMC

Ye X, Chen Y, Zhu X, Guo J, Xie D, Hou Z, Xu S, Zhou J, Fang L, Wang D, Xiao S. Cross-species transmission of deltacoronavirus and the origin of porcine deltacoronavirus. Evol Appl. 2020 Jul 31;13(9):2246-53. PubMed PMC

Zhang H, Liang Q, Li B, Cui X, Wei X, Ding Q, Wang Y, Hu H. Prevalence, phylogenetic and evolutionary analysis of porcine deltacoronavirus in Henan province, China. Prev Vet Med. 2019 May 1;166:8-15. PubMed PMC

Zheng L, Zhu J, Wang P, Shu Y, Liang Q, Li B, Wang C, Wei Z. [Establishment and preliminary application of TaqMan fluorescent quantitative RT-PCR for detection of porcine δ coronavirus]. Acta Vet Zootech Sinica. 2019 Oct;50(6):1261-7. Chinese.