Characterization and pathogenic evaluation of a novel S-INDEL PEDV CH/JSHA2024 isolated in China

Abstract

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic coronavirus of significant veterinary importance, induces severe watery diarrhea and dehydration in swine populations, with mortality rates approaching 100% in neonatal piglets. Among PEDV variants, S-INDEL strains have drawn increasing attention because of their genetic divergence and uncertain pathogenic potential in the field. In 2024, a novel S-INDEL PEDV strain, designated PEDV CH/JSHA2024, was isolated from intestinal samples of diarrheic piglets on a commercial swine farm in Jiangsu Province, China. Recombination analysis revealed that the spike (S) glycoprotein gene of this strain originated from genetic recombination between the Ch/HNLH/2015 and SQ2014 progenitor strains. Comparative genomic analysis with the prototype OH851 strain revealed multiple amino acid substitutions and insertions, including multiple amino acid substitutions and insertions within the S1 subunit, along with the absence of a conserved N-glycosylation site at position 114 (N114). The pathogenic potential of PEDV CH/JSHA2024 was assessed in pigs of different ages and maternal antibody levels. The strain caused 100% mortality in 1-day-old piglets (6/6), 50% mortality in 3-day-old piglets lacking maternal antibodies (3/6), and no mortality in 3-day-old piglets with maternal antibodies (0/6). In older animals, including 4-week-old weaned piglets and gilts, infection led to acute diarrhea and reduced feed intake but not fatality. Notably, high levels of serum IgA antibodies persisted for at least two months postinfection. These findings advance our understanding of coronavirus evolution through genetic recombination events. The establishment of this experimental model provides a valuable platform for elucidating the molecular determinants underlying S-INDEL strain pathogenesis, with particular implications for vaccine development and herd immunity strategies.

Keywords

Porcine epidemic diarrhea virus / Coronavirus / Continuous mutation / Pathogenicity / Isolation

References

[1] Boniotti, M.B., Papetti, A., Lavazza, A., Alborali, G., Sozzi, E., Chiapponi, virus and discovery of a recombinant swine enteric coronavirus, Italy. Emerg Infect Dis. 2016;22, 83–7. https://doi.org/10.3201/eid2201.150544.

[2] Bridgen, A., Kocherhans, R., Tobler, K., Carvajal, A. and Ackermann, M., 1998. Further analysis of the genome of porcine epidemic diarrhoea virus. Adv Exp Med Biol. 440, 781–6. https://doi.org/10.1007/978-1-4615-5331-1_101.

[3] Chang, S. H., J. L. Bae, T. J. Kang, J. Kim, G. H. Chung, C. W. Lim, H. Laude, M. S. Yang, and Y. S. Jang. 2002. Identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus. Molecules and Cells 14:295–299. https://doi.org/10.1016/S1016-8478(23)15106-5.

[4] Chen, J., Wang, C., Shi, H., Qiu, H., Liu, S., Chen, X., Zhang, Z. and Feng, L., 2010. Molecular epidemiology of porcine epidemic diarrhea virus in China. Arch Virol. 155, 1471–6. https://doi.org/10.1007/s00705-010-0720-2.

[5] CimaG. PED virus reinfecting U.S. herds. Virus estimated to have killed 7 million-plus pigs. Journal of the American Veterinary Medical Association, 2014, 245: 166-167

[6] Cruz, D.J., Kim, C.J. and Shin, H.J., 2008. The GPRLQPY motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize Porcine epidemic diarrhea virus. Virus Res. 132, 192–6. https://doi.org/10.1016/j.virusres.2007.10.015.

[7] Cui, M., Shen, B., Fu, Z.F. and Chen, H., 2022. Animal diseases and human future. Animal Diseases. 2, 6. https://doi.org/10.1186/s44149-022-00041-z.

[8] Gao, Q., Zheng, Z., Wang, H., Yi, S., Zhang, G. and Gong, L., 2021a. The New porcine epidemic diarrhea virus outbreak may mean that existing commercial vaccines are not enough to fully protect against the epidemic strains. Front Vet Sci. 8, 697839. https://doi.org/10.3389/fvets.2021.697839.

[9] Grasland, B., Bigault, L., Bernard, C., Quenault, H., Toulouse, O., Fablet, C., Rose, N., Touzain, F. and Blanchard, Y., 2015. Complete genome sequence of a porcine epidemic diarrhea s gene indel strain isolated in france in december 2014. Genome Announc. 3. https://doi.org/10.1128/genomeA.00535-15.

[10] Guscetti, F., C. Bernasconi, K. Tobler, K. Van Reeth, A. Pospischil, and M. Ackermann. 1998. Immunohistochemical detection of porcine epidemic diarrhea virus compared to other methods. Clinical and Diagnostic Laboratory Immunology. 5:412–414. https://doi.org/10.1128/cdli.5.3.412-414.1998.

[11] Hou, Y., Lin, C.M., Yokoyama, M., Yount, B.L., Marthaler, D., Douglas, A.L., Ghimire, S., Qin, Y., Baric, R.S., Saif, L.J. and Wang, Q., 2017. Deletion of a 197-amino-acid region in the N-terminal domain of spike protein attenuates porcine epidemic diarrhea virus in piglets. J Virol. 91. https://doi.org/10.1128/jvi.00227-17.

[12] Huang, Y.W., Dickerman, A.W., Piñeyro, P., Li, L., Fang, L., Kiehne, R., Opriessnig, T. and Meng, X.J., 2013. Origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the United States. mBio. 4, e00737–13. https://doi.org/10.1128/mBio.00737-13.

[13] Huang, C.Y., Draczkowski, P., Wang, Y.S., Chang, C.Y., Chien, Y.C., Cheng, Y.H., Wu, Y.M., Wang, C.H., Chang, Y.C., Chang, Y.C., Yang, T.J., et al. 2022. In situ structure and dynamics of an alphacoronavirus spike protein by cryo-ET and cryo-EM. Nature Communications. 13, 4877. https://doi.org/10.1038/s41467-022-32588-3.

[14] Jung, K., Saif, L.J. and Wang, Q., 2020. Porcine epidemic diarrhea virus (PEDV): An update on etiology, transmission, pathogenesis, and prevention and control. Virus Res. 286, 198045. https://doi.org/10.1016/j.virusres.2020.198045.

[15] Kocherhans, R., Bridgen, A., Ackermann, M. and Tobler, K., 2001. Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence. Virus Genes. 23, 137–44. https://doi.org/10.1023/a:1011831902219.

[16] Kuo, C.W., Yang, T.J., Chien, Y.C., Yu, P.Y., Hsu, S.D. and Khoo, K.H., 2022. Distinct shifts in site-specific glycosylation pattern of SARS-CoV-2 spike proteins associated with arising mutations in the D614G and Alpha variants. Glycobiology. 32, 60–72. https://doi.org/10.1093/glycob/cwab102.

[17] Lazov, C.M., Papetti, A., Belsham, G.J., Bøtner, A., Rasmussen, T.B. and Boniotti, M.B., 2023. Multiplex real-time RT-PCR assays for detection and differentiation of porcine enteric coronaviruses. Pathogens (Basel, Switzerland). 12. https://doi.org/10.3390/pathogens12081040.

[18] Lee, S. and Lee, C., 2014. Outbreak-related porcine epidemic diarrhea virus strains similar to US strains, South Korea, 2013. Emerg Infect Dis. 20, 1223–6. https://doi.org/10.3201/eid2007.140294.

[19] Lee, D.K., Park, C.K., Kim, S.H. and Lee, C., 2010. Heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in Korea. Virus Res. 149, 175–82. https://doi.org/10.1016/j.virusres.2010.01.015.

[20] Lee, C., 2015. Porcine epidemic diarrhea virus: An emerging and re-emerging epizootic swine virus. Virol J. 12, 193. https://doi.org/10.1186/s12985-015-0421-2.

[21] Li, R., Qiao, S., Yang, Y., Guo, J., Xie, S., Zhou, E. and Zhang, G., 2016a. Genome sequencing and analysis of a novel recombinant porcine epidemic diarrhea virus strain from Henan, China. Virus Genes. 52, 91–8. https://doi.org/10.1007/s11262-015-1254-1.

[22] Li, W., van Kuppeveld, F.J.M., He, Q., Rottier, P.J.M. and Bosch, B.J., 2016b. Cellular entry of the porcine epidemic diarrhea virus. Virus Res. 226, 117–127. https://doi.org/10.1016/j.virusres.2016.05.031.

[23] Li, C., Li, W., Lucio de Esesarte, E., Guo, H., van den Elzen, P., Aarts, E., van den Born, E., Rottier, P.J.M. and Bosch, B.J., 2017. Cell attachment domains of the porcine epidemic diarrhea virus spike protein are key targets of neutralizing antibodies. J Virol. 91. https://doi.org/10.1128/jvi.00273-17.

[24] Lin, C.M., Annamalai, T., Liu, X., Gao, X., Lu, Z., El-Tholoth, M., Hu, H., Saif, L.J. and Wang, Q., 2015. Experimental infection of a US spike-insertion deletion porcine epidemic diarrhea virus in conventional nursing piglets and cross-protection to the original US PEDV infection. Vet Res. 46, 134. https://doi.org/10.1186/s13567-015-0278-9.

[25] Liu, C., Tang, J., Ma, Y., Liang, X., Yang, Y., Peng, G., Qi, Q., Jiang, S., Li, J., Du, L. and Li, F., 2015. Receptor usage and cell entry of porcine epidemic diarrhea coronavirus. J Virol. 89, 6121–5. https://doi.org/10.1128/jvi.00430-15.

[26] López-Figueroa, C., Cano, E., Navarro, N., Pérez-Maíllo, M., Pujols, J., Núñez, J.I., Vergara-Alert, J. and Segalés, J., 2023. Clinical, pathological and virological outcomes of tissue-homogenate-derived and cell-adapted strains of porcine epidemic diarrhea virus (PEDV) in a neonatal pig model. Viruses. 16. https://doi.org/10.3390/v16010044.

[27] Machado, G., Vilalta, C., Recamonde-Mendoza, M., Corzo, C., Torremorell, M., Perez, A. and VanderWaal, K., 2019. Identifying outbreaks of Porcine Epidemic Diarrhea virus through animal movements and spatial neighborhoods. Sci Rep. 9, 457. https://doi.org/10.1038/s41598-018-36934-8.

[28] Masuda, T., Murakami, S., Takahashi, O., Miyazaki, A., Ohashi, S., Yamasato, H. and Suzuki, T., 2015. New porcine epidemic diarrhoea virus variant with a large deletion in the spike gene identified in domestic pigs. Arch Virol. 160, 2565–8. https://doi.org/10.1007/s00705-015-2522-z.

[29] Nurk, S., Bankevich, A., Antipov, D., Gurevich, A.A., Korobeynikov, A., Lapidus, A., Prjibelski, A.D., Pyshkin, A., Sirotkin, A., Sirotkin, Y., et al. 2013. Assembling single-cell genomes and mini-metagenomes from chimeric MDA products. J Comput Biol. 20, 714–37. https://doi.org/10.1089/cmb.2013.0084.

[30] Okda, F.A., Lawson, S., Singrey, A., Nelson, J., Hain, K.S., Joshi, L.R., Christopher-Hennings, J., Nelson, E.A. and Diel, D.G., 2017. The S2 glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes. Virology. 509, 185–194. https://doi.org/10.1016/j.virol.2017.06.013.

[31] Park, J.-E., 2024. Porcine Epidemic Diarrhea: Insights and progress on vaccines. Vaccines. 12. https://doi.org/10.3390/vaccines12020212.

[32] Sato, T., Takeyama, N., Katsumata, A., Tuchiya, K., Kodama, T. and Kusanagi, K., 2011. Mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo. Virus Genes. 43, 72–8. https://doi.org/10.1007/s11262-011-0617-5.

[33] Shi, K., Zhou, H., Feng, S., He, J., Li, B., Long, F., Shi, Y., Yin, Y. and Li, Z., 2023. Development of a ouadruplex RT-qPCR for the detection of porcine rotaviruses and the phylogenetic analysis of porcine RVH in China. Pathogens (Basel, Switzerland). 12. https://doi.org/10.3390/pathogens12091091.

[34] Song, D. and Park, B., 2012. Porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines. Virus Genes. 44, 167–75. https://doi.org/10.1007/s11262-012-0713-1.

[35] Song, D., Moon, H. and Kang, B., 2015. Porcine epidemic diarrhea: a review of current epidemiology and available vaccines. Clin Exp Vaccine Res. 4, 166–76. https://doi.org/10.7774/cevr.2015.4.2.166.

[36] Song, W., Feng, Y., Zhang, J., Kong, D., Fan, J., Zhao, M., Hua, L., Xiang, J., Tang, X., Xiao, S., Peng, Z. and Wu, B., 2024a. Development of a multiplex reverse transcription-quantitative PCR (qPCR) method for detecting common causative agents of swine viral diarrhea in China. Porcine Health Management. 10, 12. https://doi.org/10.1186/s40813-024-00364-y.

[37] Song, X., Li, Y., Wang, C., Zhao, Y., Yang, S., Guo, R., Hu, M., Sun, M., Zhang, G., Li, Y.,et al. 2024b. Efficacy evaluation of a bivalent subunit vaccine against epidemic PEDV heterologous strains with low cross-protection. Journal of Virology. 98, e0130924. https://doi.org/10.1128/jvi.01309-24.

[38] Stadler, J., Zoels, S., Fux, R., Hanke, D., Pohlmann, A., Blome, S., Weissenböck, H., Weissenbacher-Lang, C., Ritzmann, M. and Ladinig, A., 2015. Emergence of porcine epidemic diarrhea virus in southern Germany. BMC Vet Res. 11, 142. https://doi.org/10.1186/s12917-015-0454-1.

[39] Stevenson, G.W., Hoang, H., Schwartz, K.J., Burrough, E.R., Sun, D., Madson, D., Cooper, V.L., Pillatzki, A., Gauger, P., Schmitt, B.J., et al. 2013. Emergence of Porcine epidemic diarrhea virus in the United States: clinical signs, lesions, and viral genomic sequences. J Vet Diagn Invest. 25, 649–54. https://doi.org/10.1177/1040638713501675.

[40] Su, M., Li, C., Qi, S., Yang, D., Jiang, N., Yin, B., Guo, D., Kong, F., Yuan, D., Feng, L. and Sun, D., 2020. A molecular epidemiological investigation of PEDV in China: Characterization of co-infection and genetic diversity of S1-based genes. Transbound Emerg Dis. 67, 1129–1140. https://doi.org/10.1111/tbed.13439.

[41] Su, M., Zheng, G., Xu, X. and Song, H., 2023. Antigen epitopes of animal coronaviruses: a mini-review. Animal Diseases. 3, 14. https://doi.org/10.1186/s44149-023-00080-0.

[42] Sun, D., Feng, L., Shi, H., Chen, J., Cui, X., Chen, H., Liu, S., Tong, Y., Wang, Y. and Tong, G., 2008. Identification of two novel B cell epitopes on porcine epidemic diarrhea virus spike protein. Vet Microbiol. 131, 73–81. https://doi.org/10.1016/j.vetmic.2008.02.022.

[43] Sun, R.Q., Cai, R.J., Chen, Y.Q., Liang, P.S., Chen, D.K. and Song, C.X., 2012. Outbreak of porcine epidemic diarrhea in suckling piglets, China. Emerg Infect Dis. 18, 161–3. https://doi.org/10.3201/eid1801.111259.

[44] Sun, M., Ma, J., Wang, Y., Wang, M., Song, W., Zhang, W., Lu, C. and Yao, H., 2015. Genomic and epidemiological characteristics provide new insights into the phylogeographical and spatiotemporal spread of porcine epidemic diarrhea virus in Asia. J Clin Microbiol. 53, 1484–92. https://doi.org/10.1128/jcm.02898-14.

[45] Tian, W., Li, D., Zhang, N., Bai, G., Yuan, K., Xiao, H., Gao, F., Chen, Y., Wong, C.C.L. and Gao, G.F., 2021. O-glycosylation pattern of the SARS-CoV-2 spike protein reveals an "O-Follow-N" rule. Cell Res. 31, 1123–1125. https://doi.org/10.1038/s41422-021-00545-2.

[46] Tsai, K.J., Deng, M.C., Wang, F.I., Tsai, S.H., Chang, C., Chang, C.Y. and Huang, Y.L., 2020. Deletion in the S1 region of porcine epidemic diarrhea virus reduces the virulence and influences the virus-neutralizing activity of the antibody induced. Viruses. 12. https://doi.org/10.3390/v12121378.

[47] Van Diep, N., Sueyoshi, M., Norimine, J., Hirai, T., Myint, O., Teh, A.P.P., Izzati, U.Z., Fuke, N. and Yamaguchi, R., 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. BMC Veterinary Research. 14, 96. https://doi.org/10.1186/s12917-018-1409-0.

[48] WangDang, XiaoShaobo, Fang and Liurong, . Porcine epidemic diarrhea in China. Virus Research: An International Journal of Molecular and Cellular Virology., 2016, 226: 7-13

[49] Wang, L., Byrum, B. and Zhang, Y., 2014. New variant of porcine epidemic diarrhea virus, United States, 2014. Emerg Infect Dis. 20, 917–9. https://doi.org/10.3201/eid2005.140195.

[50] Watanabe, Y., Allen, J.D., Wrapp, D., McLellan, J.S. and Crispin, M., 2020. Site-specific glycan analysis of the SARS-CoV-2 spike. Science. 369, 330–333. https://doi.org/10.1126/science.abb9983.

[51] Won, H., Lee, D.U., Jang, G., Noh, Y.H., Lee, S.C., Choi, H.W., Yoon, I.J., Yoo, H.S. and Lee, C., 2019. Generation and protective efficacy of a cold-adapted attenuated genotype 2b porcine epidemic diarrhea virus. Journal of Veterinary Science. 20, e32. https://doi.org/10.4142/jvs.2019.20.e32.

[52] Woo, P.C., Lau, S.K., Lam, C.S., Lau, C.C., Tsang, A.K., Lau, J.H., Bai, R., Teng, J.L., Tsang, C.C., Wang, M., Zheng, B.J., Chan, K.H. and Yuen, K.Y., 2012. 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. 86, 3995–4008. https://doi.org/10.1128/jvi.06540-11.

[53] Wood, E.N., 1977. An apparently new syndrome of porcine epidemic diarrhoea. Vet Rec. 100, 243–4. https://doi.org/10.1136/vr.100.12.243.

[54] Wrapp, D. and McLellan, J.S., 2019. The 3.1-angstrom Cryo-electron microscopy structure of the porcine epidemic diarrhea virus spike protein in the prefusion conformation. J Virol. 93. https://doi.org/10.1128/jvi.00923-19.

[55] Wrapp, D., Wang, N., Corbett, K.S., Goldsmith, J.A., Hsieh, C.L., Abiona, O., Graham, B.S. and McLellan, J.S., 2020. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 367, 1260–1263. https://doi.org/10.1126/science.abb2507.

[56] Yuan, M., Wu, N.C., Zhu, X., Lee, C.D., So, R.T.Y., Lv, H., Mok, C.K.P. and Wilson, I.A., 2020. A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV. Science. 368, 630–633. https://doi.org/10.1126/science.abb7269.

[57] Zeng, Z., Li, T.T., Jin, X., Peng, F.H., Song, N.H., Peng, G.Q. and Ge, X.Y., 2017. Coexistence of multiple genotypes of porcine epidemic diarrhea virus with novel mutant S genes in the Hubei Province of China in 2016. Virol Sin. 32, 298–306. https://doi.org/10.1007/s12250-017-4021-8.

[58] Zhang, Y., Tian, Y., Lin, S.L., Sun, S.F., Chen, J., Wang, G.S., Tian, F.L. and Jiang, S.J., 2017. Two distinct genotypes of porcine epidemic diarrhoea virus in vaccinated pig flocks in Shandong Province of China, 2012–2015. Transbound Emerg Dis. 64, 1549–1556. https://doi.org/10.1111/tbed.12546.

[59] Zhang, F., Luo, Y., Lin, C., Tan, M., Wan, P., Xie, B., Xiong, L. and Ji, H., 2024. Epidemiological monitoring and genetic variation analysis of pathogens associated with porcine viral diarrhea in southern China from 2021 to 2023. Frontiers In Microbiology. 15, 1303915. https://doi.org/10.3389/fmicb.2024.1303915.

[60] Zhao, Y., Zhang, T., Zhou, C., Ma, P., Gu, K., Li, H., Li, W., Yang, X. and Wang, H., 2023. Development of an RT-PCR-based RspCas13d system to detect porcine deltacoronavirus. Applied Microbiology and Biotechnology. 107, 5739–5747. https://doi.org/10.1007/s00253-023-12690-2.

[61] Zhou, P., Fan, H., Lan, T., Yang, X.-L., Shi, W.-F., Zhang, W., Zhu, Y., Zhang, Y.-W., Xie, Q.-M., Mani, S., et al, 2018. Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat origin. Nature. 556, 255–258. https://doi.org/10.1038/s41586-018-0010-9.

[62] Zuo, Q., Zhao, R., Liu, J., Zhao, Q., Zhu, L., Zhang, B., Bi, J., Yang, G., Liu, J. and Yin, G., 2018. Epidemiology and phylogeny of spike gene of porcine epidemic diarrhea virus from Yunnan, China. Virus Res. 249, 45–51. https://doi.org/10.1016/j.virusres.2018.03.008.

Funding

National Key Research and Development Program(2021YFD1801101)

Guangdong S&T program(2023B1212070018)

Yunfu S&T program(2024020203)

RIGHTS & PERMISSIONS

The Author(s)