Abstract Abstract Porcine delta coronavirus (PDCoV) is a newly emerging swine intestinal coronavirus. In this study, a reverse transcription polymerase chain reaction (RT-PCR) method based on M gene for detecting PDCoV was developed and clinically used for epidemiology survey of PDCoV in Sichuan province and detecting the tissue distribution of PDCoV in challenged piglets. Based on the results of PDCoV M gene sequence multiple alignment and comparison with M gene sequences of Porcine epidemic diarrhea virus (PEDV) and Porcine transmissible gastroenteritis virus (TGEV), a pair of specific primers was designed and the RT-PCR (reverse transcription PCR) method for detecting PDCoV was established by determining the optimum reaction conditions and the specificity, sensitivity and reproducibility was evaluated, respectively, epidemiological investigation of PDCoV in Sichuan was conducted by detecting 226 clinical diarrhea samples, and the tissue distribution of PDCoV in infected piglets after challenging 7-day-old piglets was assessed. The results showed the target M gene fragment was 654 bp by RT-PCR, the optimum annealing temperature is 60 ℃, the upstream and downstream primer volume was 1.75 μL.The method had high sensitivity and specificity with a limited detection of 8.82×109 IU/mL for PDCoV, respectively, and no cross-reaction with other reference virus such as PEDV, TGEV, Japanese encephalitis virus (JEV), Porcine rota virus (RV), Porcine respiratory and reproductive syndrome virus (PRRSV) or Classical swine fever virus (CSFV). The positive rate of PDCoV in 226 clinical samples from Sichuan was 7.1% (16/226). The M gene of PDCoV CHN-SC2015 strain was sequenced and compared with other 28 reference M genes of PDCoV strains collected from GenBank. The results showed that the nucleotide and amino similarity was 98%~100% and 99%~100%, respectively. From the challenged piglets, PDCoV could be detected from heart, liver, spleen, lung, kidney, colon, ileum and jejunum, indicating that PDCoV had a broad tissue tropism. The established RT-PCR in this study is a simple, rapid and specific assay suitable for clinically detecting PDCoV.
LIU Gao-Yu,HUANG Xiao-Bei,LI Cheng, et al. Establishment and Application of RT-PCR Detecting Porcine deltacoronavirus (PDCoV)[J]. , 2018, 26(9): 1631-1638.
[1]Chen, JF, Sun, DB, Wang, CB, Shi, HY, Cui, XC, Liu, SW, Qiu, HJ, and Feng, L (2008): Molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in China.Virus Genes 36, 355.
[2]Chen, Q, Gauger, P, Stafne, M, Thomas, J, Arruda, P, Burrough, E, Madson, D, Brodie, J, Magstadt, D, and Derscheid, R (2015): Pathogenicity and pathogenesis of a United States porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets.Virology 482, 51-59.
[3]Hu, H (2015): Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States.Journal of Clinical Microbiology 53, 1537-48.
[4]Hu, H, Jung, K, Vlasova, AN, and Saif, LJ (2016): Experimental infection of gnotobiotic pigs with the cell-culture-adapted porcine deltacoronavirus strain OH-FD22.Archives of Virology 161, 3421.
[5]Janetanakit, T, Lumyai, M, Bunpapong, N, Boonyapisitsopa, S, Chaiyawong, S, Nonthabenjawan, N, Kesdaengsakonwut, S, and Amonsin, A (2016): Porcine Deltacoronavirus, Thailand, 2015.Emerging Infectious Diseases 22, 757-759.
[6]Jinghui, F, and Yijing, L (2005): Cloning and sequence analysis of the M gene of porcine epidemic diarrhea virus LJB/03.Virus Genes 30, 69.
[7]Jung, K, Hu, H, Eyerly, B, Lu, Z, Chepngeno, J, and Saif, LJ (2015): Pathogenicity of 2 Porcine Deltacoronavirus Strains in Gnotobiotic Pigs.Emerging Infectious Diseases 21, 650-4.
[8]Lau, SK, Tsang, CC, and Zheng, B Discovery of Seven Novel Mammalian and Avian Coronaviruses in the Genus Deltacoronavirus Supports Bat Coronaviruses as the Gene Source of Alphacoronavirus and Betacoronavirus and A.
[9]Lee, S, and Lee, C (2014): Complete Genome Characterization of Korean Porcine Deltacoronavirus Strain KOR/KNU14-04/2014.Genome Announcements 2.
[10]Ma, Y, Zhang, Y, Liang, X, Lou, F, Oglesbee, M, Krakowka, S, and Li, J (2015): Origin, evolution, and virulence of porcine deltacoronaviruses in the United States.mBio 6, e00064.
[11]Marthaler, D, Raymond, L, Jiang, Y, Collins, J, Rossow, K, and Rovira, A (2014): Rapid detection, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus.Emerging Infectious Diseases 20, 1347-50.
[12]Nan, D, Liurong, F, Songlin, Z, Qianqian, S, Huanchun, C, and Shaobo, X (2015): Porcine Deltacoronavirus in Mainland China.Emerging Infectious Diseases 21, 2254.
[13]Sinha, A, Gauger, P, Zhang, J, Yoon, KJ, and Harmon, K (2015): PCR-based retrospective evaluation of diagnostic samples for emergence of porcine deltacoronavirus in US swine.Veterinary Microbiology 179, 296-298.
[14]Siu, KL, Kok, KH, Ng, MH, Poon, VK, Yuen, KY, Zheng, BJ, and Jin, DY (2009): Severe acute respiratory syndrome coronavirus M protein inhibits type I interferon production by impeding the formation of TRAF3.TANK.TBK1/IKKepsilon complex. Journal of Biological Chemistry 284, 16202-9.
[15]Song, D, Zhou, X, Peng, Q, Chen, Y, Zhang, F, Huang, T, Zhang, T, Li, A, Huang, D, and Wu, Q (2015): Newly Emerged Porcine Deltacoronavirus Associated With Diarrhoea in Swine in China: Identification, Prevalence and Full-Length Genome Sequence Analysis.Transboundary and emerging diseases 62, 575.
[16]Thachil, A, Gerber, PF, Xiao, CT, Huang, YW, and Opriessnig, T (2015): Development and Application of an ELISA for the Detection of Porcine Deltacoronavirus IgG Antibodies.Plos One 10, e0124363.
[17]Wang, L, Byrum, B, and Zhang, Y (2014): Porcine Coronavirus HKU15 Detected in 9 US States, 2014.Emerging Infectious Diseases 20, 1594-5.
[18]Zhai, SL, Wei, WK, Li, XP, Wen, XH, Zhou, X, Zhang, H, Lv, DH, Li, F, and Wang, D (2016): Occurrence and sequence analysis of porcine deltacoronaviruses in southern China.Virology Journal 13, 136.
[19]方谱县, 方六荣, 董楠, and 肖少波 (2016): 猪δ冠状病毒的研究进展.病毒学报, 243-248.
