|
|
|
| Preparation and Identification of Monoclonal Antibodies Distinguishing Porcine epidemic diarrhea virus Variants from Classical Strains |
| WANG Ya-Wen1,*, ZHAO Xue1,*, LI Tan-Qing1, MI Jian-Hua2, MIAO Yun-Yan2, SUN Li-Dan1, ZHANG Yi-Ming1, SONG Qin-Ye1,* |
1 Research Center of Veterinary Biologicals Engineering and Technology of Hebei/College of Veterinary Medicine, Hebei Agricultural University, Baoding 071000, China;
2 Hebei Laishui County Animal Husbandry and Aquaculture Bureau, Baoding 074199, China |
|
|
|
|
Abstract Porcine epidemic diarrhea (PED) caused by Porcine epidemic diarrhea virus (PEDV) is a highly contagious and devastating enteric infectious disease to pig (Sus scrofa) production. Serological techniques are absent for distinguishing PEDV variants from classical strains. Monoclonal antibody (McAb) is a kind of important and basic experimental materials for serological testing or diagnosing technique researches. In order to prepare McAb for distinguishing PEDV variants from classical strains, this study compared and analyzed the nucleotide and amino acid sequences of spike (S) protein of PEDV virulent variants and classical strains by the software of DNAMAN and DANStar7.1. The polypeptide, 55IGENQGVNSTWYCAGRHPTAS75 being different between the PEDV variants and the classical ones, was screened out and synthesized. BALB/c mice (Mus musculus) were immunized 3 times at an interval of 2 weeks with the polypeptide conjugated keyhole limpet haemocyanin (KLH), and then one strain of hybridoma (named 3#) which secreted IgG2bκ monoclonal antibody (named McAb 3#) was obtained through cell fusion technique, 3 rounds of clone selecting as well as enzyme-linked immunosorbent assay (ELISA) detection. The McAb could specifically react with PEDV-S protein whereas it did not combine with PEDV nucleocapsid (N) protein by ELISA and Western blot, meanwhile, PEDV variants could specifically bind to the McAb 3# in Vero-81 cells using the indirect immunofluorescence assay (IFA). The specific ELISA antibody titer reached to 1∶106 in mouse ascitic fluid induced by 3# hybridoma cells. Moreover, IFA based on the McAb 3# was performed in antigen detection of PEDV variant HBQY2016 and the classical attenuated strain CV777 in Vero-81 cells, respectively. Green fluorescence signals were observed only in the cytoplasm of Vero-81 cells infected with PEDV variant HBQY2016, but not in the cells infected with the classical attenuated strain CV777, which indicated the McAb 3# could distinguish the PEDV variant from the classical strain. The results showed that the McAb prepared in this study specifically combined with PEDV S protein and could distinguish the PEDV variant from classical strain. Its preparation could be used for studying PEDV infection immunity and developing antigen or antibody detection methods to differentiate PEDV variants from classical strains.
|
|
Received: 18 December 2019
|
|
|
|
Corresponding Authors:
**, songqinye@126.con
|
| About author:: * The authors who contribute equally |
|
|
|
[1] 张庆桥, 王一鹏, 魏艳秋, 等. 2019. 猪流行性腹泻N蛋白单克隆抗体的制备与鉴定[J]. 中国兽医杂志, 55(1): 31-35.
(Zhang Q Q, Wang Y P, Wei Y Q, et al.2019. Preparation and identification of monoclonal antibodies against N protein of Porcine epidemic diarrhea virus[J]. Chinese Journal of Veterinary Medicine, 55(1): 31-35.
[2] 朱卫霞, 郭海勇, 陈立功, 等. 2016. 猪流行性腹泻病毒HBMC2012 株的分离鉴定及其致病性研究[J]. 中国预防兽医学报, 38(12): 934-938.
(Zhu W X, Guo H Y, Chen L G, et al.2016. Isolation and identification of Porcine epidemic diarrhea virus strain HBMC2012 and its pathogenicity[J]. Chinese Journal of Preventive Veterinary Medicine, 38(12): 934-938.)
[3] Antas M, Woźniakowski G.2019. Current status of porcine epidemic diarrhoea (PED) in European pigs[J]. Journal of Veterinary Research, 63(4): 465-470.
[4] Chang S H, Bae J L, Kang T J, et al.2002. Identification of the epitope region capable of inducing neutralizing antibodies against the Porcine epidemic diarrhea virus[J]. Molecules and Cells, 14(2): 295-299.
[5] Chen J F, Sun D B, Wang C B, et al.2008. Molecular characterization and phylogenetic analysis of membrane protein genes of Porcine epidemic diarrhea virus isolates in China[J]. Virus Genes, 36(2): 355-364.
[6] Chen J, Wang C, Shi H, et al.2010. Molecular epidemiology of Porcine epidemic diarrhea virus in China[J]. Archives of Virology, 155(9): 1471-1476.
[7] Chen N, Li S, Zhou R, et al.2017. Two novel Porcine epidemic diarrhea virus (PEDV) recombinants from a natural recombinant and distinct subtypes of PEDV variants[J]. Virus Research, 242: 90-95.
[8] Chen Q, Gauger P C, Stafne M R, et al.2016. Pathogenesis comparison between the United States Porcine epidemic diarrhoea virus prototype and S-INDEL-variant strains in conventional neonatal piglets[J]. Journal of General Virology, 97(5): 1107-1121.
[9] Choudhury B, Dastjerdi A, Doyle N, et al.2016. From the field to the lab-An European view on the global spread of PEDV[J]. Virus Research, 226: 40-49.
[10] Cruz D J, Kim C J, Shin H J.2008. The GPRLQPY motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize Porcine epidemic diarrhea virus[J]. Virus Research, 132(1-2): 192-196.
[11] Lee C.2016. Porcine epidemic diarrhea virus: An emerging and re-emerging epizootic swine virus[J]. Virology Journal, 13: 19.
[12] Li W, Li H, Liu Y, et al.2012. New variants of Porcine epidemic diarrhea virus, China, 2011[J]. Emerging Infectious Diseases, 18(8): 1350-1353.
[13] Liu J, Li L, Han J, et al.2019. A TaqMan probe-based real-time PCR to differentiate Porcine epidemic diarrhea virus virulent strains from attenuated vaccine strains[J]. Molecular and Cellular Probes, 45: 37-42.
[14] Okda F, Liu X, Singrey A, et al.2015. Development of an indirect ELISA, blocking ELISA, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to North American strains of Porcine epidemic diarrhea virus[J]. BMC Veterinary Research, 11: 180.
[15] Pan X, Kong N, Shan T, et al.2015. Monoclonal antibody to N protein of Porcine epidemic diarrhea virus[J]. Monoclonal Antibodies Immunodiagnosis Immunotherapy, 34(1): 51-54.
[16] Saif L J, Pensaert M B, Sestack K, et al.2012. Coronaviruses. In: Straw B E, Zimmerman J J, Karriker L A, et al., editors Diseases of Swine[J]. Ames: Wiley-Blackwell, pp. 501-524.
[17] Song D, Huang D, Peng Q, et al.2015. Molecular characterization and phylogenetic analysis of Porcine epidemic diarrhea viruses associated with outbreaks of severe diarrhea in piglets in Jiangxi, China 2013[J]. The Public Liabrary of Science One, 10(3): e0120310.
[18] Song D, Park B.2012. Porcine epidemic diarrhoea virus: A comprehensive review of molecular epidemiology, diagnosis, and vaccines[J]. Virus Genes, 44(2): 167-175.
[19] Song Q, Stone S, Drebes D, et al.2016. Characterization of anti-Porcine epidemic diarrhea virus neutralizing activity inmammary secretions[J]. Virus Research, 226: 85-92.
[20] Sozzi E, Moreno A, Lelli D, et al.2018. Development and validation of a monoclonal antibody-based competitive ELISA for detection of antibodies against Porcine epidemic diarrhoea virus (PEDV)[J]. Research in Veterinary Science, 121: 106-110.
[21] Su Y, Hou Y, Prarat M, et al.2018a. New variants of Porcine epidemic diarrhea virus with large deletions in the spike protein, identified in the United States, 2016-2017[J]. Archives of Virology, 163(9): 2485-2489.
[22] Su Y, Liu Y, Chen Y, et al.2018b. A novel duplex TaqMan probe-based real-time RT-qPCR for detecting and differentiating classical and variant Porcine epidemic diarrhea viruses[J]. Molecular and Cellular Probes, 37: 6-11.
[23] Sun D, Feng L, Shi H, et al.2008. Identification of two novel B cell epitopes on Porcine epidemic diarrhea virus spike protein[J]. Veterinary Microbiology, 131(1-2): 73-81.
[24] Sun D, Wang X, Wei S, et al.2016. Epidemiology and vaccine of Porcine epidemic diarrhea virus in China: A mini-review[J]. Journal of Veterinary Medical Science, 78(3): 355-363.
[25] Suzuki T, Shibahara T, Yamaguchi R, et al.2016. Pig epidemic diarrhoea virus S gene variant with a large deletion non-lethal to colostrum-deprived newborn piglets[J]. Journal of General Virology, 97(8): 1823-1828.
[26] van Diep N, Sueyoshi M, Norimine J, et al.2018. Molecular characterization of US-like and Asian non-S INDEL strains of Porcine epidemic diarrhea virus (PEDV) that circulated in Japan during 2013-2016 and PEDVs collected from recurrent outbreaks[J]. BMC Veterinary Research, 14(1): 96.
[27] Wang E, Guo D, Li C, et al.2016. Molecular characterization of the ORF3 and S1 genes of Porcine epidemic diarrhea virus non S-INDEL strains in seven regions of China, 2015[J]. The Public Liabrary of Science One, 11(8): e0160561.
[28] Wang X M, Niu B B, Yan H, et al.2013. Genetic properties of endemic Chinese Porcine epidemic diarrhea virus strains isolated since 2010[J]. Archives of Virology, 158(12): 2487-2494.
[29] Zhang F, Luo S, Gu J, et al.2019. Prevalence and phylogenetic analysis of porcine diarrhea associated viruses in southern China from 2012 to 2018[J]. BMC Veterinary Research, 15(1): 470.
[30] Zhang Q, Liu X, Fang Y, et al.2017. Detection and phylogenetic analyses of spike genes in Porcine epidemic diarrhea virus strains circulating in China in 2016-2017[J]. Virology Journal, 14(1): 194.
[31] Zhao P, Bai J, Jian g, et al.2014. Development of a multiplex TaqMan probe-based real-time PCR for discrimination of variant and classical Porcine epidemic diarrhea virus[J]. Journal of Virological Methods, 206: 150-155. |
|
|
|
